Glycosylated interferon α

ABSTRACT

This invention provides for human interferon-α which includes novel glycosylation structures.

This application is a continuation of U.S. application Ser. No.10/351,196, filed Jan. 24, 2003, now U.S. Pat. No. 7,129,390, issuedOct. 31, 2006, the disclosure of which is incorporated in its entiretyherein by reference.

BACKGROUND OF THE INVENTION

a) Field of the Invention

The present invention relates to vectors and methods for theintroduction of exogenous genetic material into avian cells and theexpression of the exogenous genetic material in the cells. The inventionalso relates to transgenic avian species, including chicken and turkey,and to avian eggs which contain exogenous protein.

b) Description of Related Art

Numerous natural and synthetic proteins are used in diagnostic andtherapeutic applications; many others are in development or in clinicaltrials. Current methods of protein production include isolation fromnatural sources and recombinant production in bacterial and mammaliancells. Because of the complexity and high cost of these methods ofprotein production, however, efforts are underway to developalternatives. For example, methods for producing exogenous proteins inthe milk of pigs, sheep, goats, and cows have been reported. Theseapproaches suffer from several limitations, including long generationtimes between founder and production transgenic herds, extensivehusbandry and veterinary costs, and variable levels of expressionbecause of position effects at the site of the transgene insertion inthe genome. Proteins are also being produced using milling and maltingprocesses from barley and rye. However, plant post-translationalmodifications differ from vertebrate post-translational modifications,which often has a critical effect on the function of the exogenousproteins.

The Oviduct as a Bioreactor

Like tissue culture and mammary gland bioreactors, the avian oviduct canalso potentially serve as a bioreactor. Successful methods of modifyingavian genetic material such that high levels of exogenous proteins aresecreted in and packaged into eggs would allow inexpensive production oflarge amounts of protein. Several advantages of such an approach wouldbe: a) short generation times (24 weeks) and rapid establishment oftransgenic flocks via artificial insemination; b) readily scaledproduction by increasing flock sizes to meet production needs; c)post-translational modification of expressed proteins; d) automatedfeeding and egg collection; e) naturally sterile egg-whites; and f)reduced processing costs due to the high concentration of protein in theegg white.

The avian reproductive system, including that of the chicken, is welldescribed. The egg of the hen consists of several layers which aresecreted upon the yolk during its passage through the oviduct. Theproduction of an egg begins with formation of the large yolk in theovary of the hen. The unfertilized oocyte is then positioned on top ofthe yolk sac. Upon ovulation or release of the yolk from the ovary, theoocyte passes into the infundibulum of the oviduct where it isfertilized if sperm are present. It then moves into the magnum of theoviduct which is lined with tubular gland cells. These cells secrete theegg-white proteins, including ovalbumin, lysozyme, ovomucoid,conalbumin, and ovomucin, into the lumen of the magnum where they aredeposited onto the avian embryo and yolk.

The ovalbumin gene encodes a 45 kD protein that is specificallyexpressed in the tubular gland cells of the magnum of the oviduct(Beato, Cell 56:335-344 (1989)). Ovalbumin is the most abundant eggwhite protein, comprising over 50 percent of the total protein producedby the tubular gland cells, or about 4 grams of protein per large GradeA egg (Gilbert, “Egg albumen and its formation” in Physiology andBiochemistry of the Domestic Fowl, Bell and Freeman, eds., AcademicPress, London, New York, pp. 1291-1329). The ovalbumin gene and over 20kb of each flanking region have been cloned and analyzed (Lai et al.,Proc. Natl. Acad. Sci. USA 75:2205-2209 (1978); Gannon et al., Nature278:428-424 (1979); Roop et al., Cell 19:63-68 (1980); and Royal et al.,Nature 279:125-132 (1975)).

Much attention has been paid to the regulation of the ovalbumin gene.The gene responds to steroid hormones such as estrogen, glucocorticoids,and progesterone, which induce the accumulation of about 70,000ovalbumin mRNA transcripts per tubular gland cell in immature chicks and100,000 ovalbumin mRNA transcripts per tubular gland cell in the maturelaying hen (Palmiter, J. Biol. Chem. 248:8260-8270 (1973); Palmiter,Cell 4:189-197 (1975)). DNAse hypersensitivity analysis andpromoter-reporter gene assays in transfected tubular gland cells defineda 7.4 kb region as containing sequences required for ovalbumin geneexpression. This 5′ flanking region contains four DNAse I-hypersensitivesites centered at −0.25, −0.8, −3.2, and −6.0 kb from the transcriptionstart site. These sites are called HS-I, -II, -III, and -IV,respectively. These regions reflect alterations in the chromatinstructure and are specifically correlated with ovalbumin gene expressionin oviduct cells (Kaye et al., EMBO 3:1137-1144 (1984)).Hypersensitivity of HS-II and -III are estrogen-induced, supporting arole for these regions in hormone-induction of ovalbumin geneexpression.

HS-I and HS-II are both required for steroid induction of ovalbumin genetranscription, and a 1.4 kb portion of the 5′ region that includes theseelements is sufficient to drive steroid-dependent ovalbumin expressionin explanted tubular gland cells (Sanders and McKnight, Biochemistry 27:6550-6557 (1988)). HS-I is termed the negative-response element (“NRE”)because it contains several negative regulatory elements which repressovalbumin expression in the absence of hormones (Haekers et al., Mol.Endo. 9:1113-1126 (1995)). Protein factors bind these elements,including some factors only found in oviduct nuclei suggesting a role intissue-specific expression. HS-II is termed the steroid-dependentresponse element (“SDRE”) because it is required to promote steroidinduction of transcription. It binds a protein or protein complex knownas Chirp-I. Chirp-I is induced by estrogen and turns over rapidly in thepresence of cyclohexamide (Dean et al., Mol. Cell. Biol. 16:2015-2024(1996)). Experiments using an explanted tubular gland cell culturesystem defined an additional set of factors that bind SDRE in asteroid-dependent manner, including a NFκB-like factor (Nordstrom etal., J. Biol. Chem. 268:13193-13202 (1993); Schweers and Sanders, J.Biol. Chem. 266:10490-10497 (1991)).

Less is known about the function of HS-III and -IV. HS-III contains afunctional estrogen response element, and confers estrogen inducibilityto either the ovalbumin proximal promoter or a heterologous promoterwhen co-transfected into HeLa cells with an estrogen receptor cDNA.These data imply that HS-III may play a functional role in the overallregulation of the ovalbumin gene. Little is known about the function ofHS-IV, except that it does not contain a functional estrogen-responseelement (Kato et al., Cell 68: 731-742 (1992)).

There has been much interest in modifying eukaryotic genomes byintroducing foreign genetic material and/or by disrupting specificgenes. Certain eukaryotic cells may prove to be superior hosts for theproduction of exogenous eukaryotic proteins. The introduction of genesencoding certain proteins also allows for the creation of new phenotypeswhich could have increased economic value. In addition, somegenetically-caused disease states may be cured by the introduction of aforeign gene that allows the genetically defective cells to express theprotein that it can otherwise not produce. Finally, modification ofanimal genomes by insertion or removal of genetic material permits basicstudies of gene function, and ultimately may permit the introduction ofgenes that could be used to cure disease states, or result in improvedanimal phenotypes.

Transgenic Animals

Transgenesis has been accomplished in mammals by several differentmethods. First, in mammals including the mouse, pig, goat, sheep andcow, a transgene is microinjected into the pronucleus of a fertilizedegg, which is then placed in the uterus of a foster mother where itgives rise to a founder animal carrying the transgene in its germline.The transgene is engineered to carry a promoter with specific regulatorysequences directing the expression of the foreign protein to aparticular cell type. Since the transgene inserts randomly into thegenome, position effects at the site of the transgene's insertion intothe genome may variably cause decreased levels of transgene expression.This approach also requires characterization of the promoter such thatsequences necessary to direct expression of the transgene in the desiredcell type are defined and included in the transgene vector (Hogan et al.Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory, NY(1988)).

A second method for effecting animal transgenesis is targeted genedisruption, in which a targeting vector bearing sequences of the targetgene flanking a selectable marker gene is introduced into embryonic stem(“ES”) cells. Via homologous recombination, the targeting vectorreplaces the target gene sequences at the chromosomal locus or insertsinto interior sequences preventing expression of the target geneproduct. Clones of ES cells bearing the appropriately disrupted gene areselected and then injected into early stage blastocysts generatingchimeric founder animals, some of which bear the transgene in the germline. In the case where the transgene deletes the target locus, itreplaces the target locus with foreign DNA borne in the transgenevector, which consists of DNA encoding a selectable marker useful fordetecting transfected ES cells in culture and may additionally containDNA sequences encoding a foreign protein which is then inserted in placeof the deleted gene such that the target gene promoter drives expressionof the foreign gene (U.S. Pat. Nos. 5,464,764 and 5,487,992 (M. P.Capecchi and K. R. Thomas)). This approach suffers from the limitationthat ES cells are unavailable in many mammals, including goats, cows,sheep and pigs. Furthermore, this method is not useful when the deletedgene is required for survival or proper development of the organism orcell type.

Recent developments in avian transgenesis have allowed the modificationof avian genomes. Germ-line transgenic chickens may be produced byinjecting replication-defective retrovirus into the subgerminal cavityof chick blastoderms in freshly laid eggs (U.S. Pat. No. 5,162,215;Bosselman et al., Science 243:533-534 (1989); Thoraval et al.,Transgenic Research 4:369-36 (1995)). The retroviral nucleic acidcarrying a foreign gene randomly inserts into a chromosome of theembryonic cells, generating transgenic animals, some of which bear thetransgene in their germ line. Use of insulator elements inserted at the5′ or 3′ region of the fused gene construct to overcome position effectsat the site of insertion has been described (Chim et al., Cell74:504-514 (1993)).

In another approach, a transgene has been microinjected into thegerminal disc of a fertilized egg to produce a stable transgenic founderavian that passes the gene to the F1 generation (Love et al.Bio/Technology 12:60-63 (1994)). However, this method has severaldisadvantages. Hens must be sacrificed in order to collect thefertilized egg, the fraction of transgenic founders is low, and injectedeggs require labor intensive in vitro culture in surrogate shells.

In another approach, blastodermal cells containing presumptiveprimordial germ cells (“PGCs”) are excised from donor eggs, transfectedwith a transgene and introduced into the subgerminal cavity of recipientembryos. The transfected donor cells are incorporated into the recipientembryos generating transgenic embryos, some of which are expected tobear the transgene in the germ line. The transgene inserts in randomchromosomal sites by nonhomologous recombination. However, no transgenicfounder avians have yet been generated by this method.

Lui, Poult. Sci. 68:999-1010 (1995), used a targeting vector containingflanking DNA sequences of the vitellogenin gene to delete part of theresident gene in chicken blastodermal cells in culture. However, it hasnot been demonstrated that these cells can contribute to the germ lineand thus produce a transgenic embryo. In addition, this method is notuseful when the deleted gene is required for survival or properdevelopment of the organism or cell type.

Thus, it can be seen that there is a need for a method of introducingforeign DNA, operably linked to a suitable promoter, into the aviangenome such that efficient expression of an exogenous gene can beachieved. Furthermore, there exists a need to create germ-line modifiedtransgenic avians which express exogenous genes in their oviducts andsecrete the expressed exogenous proteins into their eggs.

Interferon

When interferon was discovered in 1957, it was hailed as a significantantiviral agent. In the late 1970s, interferon became associated withrecombinant gene technology. Today, interferon is a symbol of thecomplexity of the biological processes of cancer and the value ofendurance and persistence in tackling this complexity.

The abnormal genes that cause cancer comprise at least three types:Firstly, there are the oncogenes, which, when altered, encourage theabnormal growth and division that characterize cancer. Secondly, thereare the tumor suppressor genes, which, when altered, fail to controlthis abnormal growth and division. Thirdly, there are the DNA repairgenes, which, when altered, fail to repair mutations that can lead tocancer. Researchers speculate that there are about 30 to 40 tumorsuppressor genes in the body, each of which produces a protein. Theseproteins may be controlled by “master” tumor suppressor proteins such asRb (for retinoblastoma, with which it was first associated) and p53(associated with many different tumors). Evidence from the laboratorysuggests that returning just one of these tumor suppressor genes to itsnormal function can appreciably reduce the aggressiveness of themalignancy.

Scientists became intrigued by interferon when it was discovered thatinterferon can inhibit cell growth. Further, interferon was found tohave certain positive effects on the immune system. It is now consideredanalogous to a tumor suppressor protein: it inhibits the growth ofcells, particularly malignant cells; it blocks the effects of manyoncogenes and growth factors; and unlike other biological agents, itinhibits cell motility which is critical to the process of metastasis.

Intercellular communication is dependent on the proper functioning ofall the structural components of the tissue through which the messagesare conveyed: the matrix, the cell membrane, the cytoskeleton, and thecell itself. In cancer, the communication network between cells isdisrupted. If the cytoskeleton is disrupted, the messages don't getthrough to the nucleus and the nucleus begins to function abnormally.Since the nucleus is the site where the oncogenes or tumor suppressorgenes get switched on or off, this abnormal functioning can lead tomalignancy. When this happens, the cells start growing irregularly anddo not differentiate. They may also start to move and disrupt othercells. It is believed that interferon, probably in concert with otherextracellular and cellular substances, restores the balance andhomeostasis, making sure the messages get through properly. Interferonstops growth, stops motility, and enhances the ability of the cell,through adhesion molecules, to respond to its environment. It alsocorrects defects and injuries in the cytoskeleton. Interferon has beenfound to block angiogenesis, the initial step in the formation of newblood vessels that is essential to the growth of malignancies. Moreover,it blocks fibrosis, a response to injury that stimulates many differentkinds of cells and promotes cell growth (Kathryn L. Hale, Oncolog,Interferon: The Evolution of a Biological Therapy, Taking a New Look atCytokine Biology).

Interferon is produced by animal cells when they are invaded by virusesand is released into the bloodstream or intercellular fluid to inducehealthy cells to manufacture an enzyme that counters the infection. Formany years the supply of human interferon for research was limited bycostly extraction techniques. In 1980, however, the protein becameavailable in greater quantities through genetic engineering (i.e.,recombinant forms of the protein). Scientists also determined that thebody makes three distinct types of interferon, referred to as α-(alpha),β-(beta), and γ-(gamma) interferon. Interferons were first thought to behighly species-specific, but it is now known that individual interferonsmay have different ranges of activity in other species. Alpha interferon(α-IFN) has been approved for therapeutic use against hairy-cellleukemia and hepatitis C. α-IFN has also been found effective againstchronic hepatitis B, a major cause of liver cancer and cirrhosis, aswell as for treatment of genital warts and some rarer cancers of bloodand bone marrow. Nasal sprays containing α-IFN provide some protectionagainst colds caused by rhinoviruses. Human α-IFN belongs to a family ofextra-cellular signaling proteins with antiviral, antiproliferating andimmunomodulatory activities. IFN-α proteins are encoded by a multigenefamily which includes 13 genes clustered on the human chromosome 9. Mostof the IFN-α genes are expressed at the mRNA level in leukocytes inducedby Sendai virus. Further, it has been shown that at least nine differentsub-types are also produced at the protein level. The biologicalsignificance of the expression of several similar IFN-α proteins is notknown, however, it is believed that they have quantitatively distinctpatterns of antiviral, growth inhibitory and killer-cell-stimulatoryactivities. Currently, two IFN-α variants, IFN-α 2a and IFN-α 2b, aremass produced in Escherichia coli by recombinant technology and marketedas drugs. Unlike natural IFN-α, these recombinant IFN-αproducts havebeen shown to be immunogenic in some patients, which could be due tounnatural forms of IFN-α proteins. Thus, for the development of IFN-αdrugs it is necessary to not only identify the IFN-α subtypes andvariants expressed in normal human leukocytes, but also to characterizetheir possible post-translational modifications (Nyman et al. (1998)Eur. J. Biochem. 253:485-493).

Nyman et al. (supra) studied the glycosylation of natural human IFN-α.They found that two out of nine of the subtypes produced by leukocytesafter a Sendai-virus induction were found to be glycosylated, namelyIFN-α 14c and IFN-α 2b, which is consistent with earlier studies. IFN-α14 is the only IFN-α subtype with potential N-glycosylation sites, Asn2and Asn72, but only Asn72 is actually glycosylated. IFN-α 2 isO-glycosylated at Threonine106 (Thr106). Interestingly, no other IFN-αsubtype contains Thr at this position. In this study, Nyman et al.liberated and isolated the oligosaccharide chains and analyzed theirstructures by mass spectrometry and specific glycosidase digestions.Both IFN-α 2b and IFN-α 14c resolved into three peaks in reversed-phasehigh performance liquid chromatography (RP-HPLC). Electrosprayionization mass spectrometry (ESI-MS) analysis of IFN-α 2b fractionsfrom RP-HPLC revealed differences in their molecular masses, suggestingthat these represent different glycoforms. This was confirmed bymasspectrometric analysis of the liberated O-glycans of each fraction.IFN-α 2b was estimated to contain about 20% of the core type-2pentasaccharide, and about 50% of disialylated and 30% of monosialylatedcore type-1 glycans. Nyman et al.'s data agrees with previous partialcharacterization of IFN-α 2b glycosylation (Adolf et al. (1991) Biochem.J. 276:511-518). The role of glycosylation in IFN-α 14c and IFN-α 2b isnot clearly established. According to Nyman et al. (supra), thecarbohydrate chains are not essential for the biological activity, butglycosylation may have an effect on the pharmacokinetics and stabilityof the proteins.

There are at least 15 functional genes in the human genome that code forproteins of the IFN-α family. The amino acid sequence similarities aregenerally in the region of about 90%, thus, these molecules are closelyrelated in structure. IFN-α proteins contain 166 amino acids (with theexception of IFN-α 2, which has 165 amino acids) and characteristicallycontain four conserved cysteine residues which form two disulfidebridges. IFN-α species are slightly acidic in character and lack arecognition site for asparagine-linked glycosylation (with the exceptionof IFN-α 14 which does contain a recognition site for asparagine-linkedglycosylation). Three variants of IFN-α 2, differing in their aminoacids at positions 23 and 34, are known: IFN-α 2a (Lys-23, His-34);IFN-α 2b (Arg-23, His-34); and IFN-α 2c (Arg-23, Arg-34). Two otherhuman IFN species, namely IFN-ω1 and IFN-β are N-glycosylated and aremore distantly related to IFN-α. IFN-α, -β and -ω, collectively referredto as class I IFNs, bind to the same high affinity cell membranereceptor (Adolf et al. (1991) Biochem. J. 276:511-518).

Adolf et al. (supra) used the specificity of a monoclonal antibody forthe isolation of natural IFN-α 2 from human leukocyte IFN. They obtaineda 95% pure protein through immunoaffinity chromatography which confirmedthe expected antiviral activity of IFN-α 2. Analysis of natural IFN-α 2by reverse-phase HPLC, showed that the natural protein can be resolvedinto two components, both more hydrophilic than E. coli-derived IFN-α 2.SDS/PAGE revealed that the protein is also heterogeneous in molecularmass, resulting in three bands, all of them with lower electrophoreticmobility than the equivalent E. coli-derived protein.

Adolf et al. (supra) also speculated that natural IFN-α 2 carriesO-linked carbohydrate residues. Their hypothesis was confirmed bycleavage of the putative peptide-carbohydrate bond with alkali; theresulting protein was homogeneous and showed the same molecular mass asthe recombinant protein. Further comparison of natural and recombinantproteins after proteolytic cleavage, followed by separation and analysisof the resulting fragments, allowed them to define a candidateglycopeptide. Sequence analysis of this peptide identified Thr-106 asthe O-glycosylation site. A comparison of the amino acid sequences ofall published IFN-α 2 species revealed that this threonine residue isunique to IFN-α 2. Glycine, isoleucine or glutamic acid are present atthe corresponding position (107) in all other proteins.

Preparations of IFN-α 2 produced in E. coli are devoid ofO-glycosylation and have been registered as drugs in many countries.However, the immunogenicity of therapeutically applied E. coli-derivedIFN-α 2 might be affected by the lack of glycosylation. Studies haveshown that four out of sixteen patients receiving recombinant humangranulocyte-macrophage colony-stimulating factor produced in yeastdeveloped antibodies to this protein. Interestingly, these antibodieswere found to react with epitopes that in the endogenousgranulocyte-macrophage colony-stimulating factor are protected byO-linked glycosylation, but which are exposed in the recombinant factor(Adolf et al., supra).

Similarly, induction of antibodies to recombinant E. coli-derived IFN-α2 after prolonged treatment of patients has been described and it hasbeen speculated that natural IFN-α 2 may be less immunogenic than therecombinant IFN-α 2 proteins (Galton et al. (1989) Lancet 2:572-573).

SUMMARY OF THE INVENTION

This invention provides vectors and methods for the stable introductionof exogenous nucleic acid sequences into the genome of avians in orderto express the exogenous sequences to alter the phenotype of the aviansor to produce desired proteins. In particular, transgenic avians areproduced which express exogenous sequences in their oviducts and whichdeposit exogenous proteins into their eggs. Avian eggs that containexogenous proteins are encompassed by this invention. The instantinvention further provides novel forms of interferon and erythropoietinwhich are efficiently expressed in the oviduct of transgenic avians anddeposited into avian eggs.

One aspect of the present invention provides methods for producingexogenous proteins in specific tissues of avians. Exogenous proteins maybe expressed in the oviduct, blood and/or other cells and tissues of theavian. Transgenes are introduced into embryonic blastodermal cells,preferably near stage X, to produce a transgenic avian, such that theprotein of interest is expressed in the tubular gland cells of themagnum of the oviduct, secreted into the lumen, and deposited into theegg white of a hard shell egg. A transgenic avian so produced carriesthe transgene in its germ line. The exogenous genes can therefore betransmitted to avians by both artificial introduction of the exogenousgene into avian embryonic cells, and by the transmission of theexogenous gene to the avian's offspring stably in a Mendelian fashion.

The present invention encompasses a method of producing an exogenousprotein in an avian oviduct. The method comprises as a first stepproviding a vector that contains a coding sequence and a promoteroperably linked to the coding sequence, so that the promoter can effectexpression of the nucleic acid in the avian oviduct. Next, transgeniccells and/or tissues are created, wherein the vector is introduced intoavian embryonic blastodermal cells, either freshly isolated, in culture,or in an embryo, so that the vector sequence is randomly inserted intothe avian genome. Finally, a mature transgenic avian which expresses theexogenous protein in its oviduct is derived from the transgenic cellsand/or tissue. This method can also be used to produce an avian eggwhich contains exogenous protein when the exogenous protein that isexpressed in the oviduct is also secreted into the oviduct lumen anddeposited into the egg white of a hard shell egg.

In one aspect, the production of a transgenic bird by random chromosomalinsertion of a vector into its avian genome may optionally involve DNAtransfection of embryonic blastodermal cells which are then injectedinto the subgerminal cavity beneath a recipient blastoderm. The vectorused in such a method has a promoter which is fused to an exogenouscoding sequence and directs expression of the coding sequence in thetubular gland cells of the oviduct.

In another aspect of the invention, a random chromosomal insertion andthe production of a transgenic avian is accomplished by transduction ofembryonic blastodermal cells with replication-defective orreplication-competent retroviral particles carrying the transgenegenetic code between the 5′ and 3′ LTRs of the retroviral rector. Forinstance, an avian leukosis virus (ALV) retroviral vector or a murineleukemia virus (MLV) retroviral vector may be used which comprises amodified pNLB plasmid containing an exogenous gene that is inserteddownstream of a segment of a promoter region. An RNA copy of themodified retroviral vector, packaged into viral particles, is used toinfect embryonic blastoderms which develop into transgenic avians.Alternatively, helper cells which produce the retroviral transducingparticles are delivered to the embryonic blastoderm.

Another aspect of the invention provides a vector which includes acoding sequence and a promoter in operational and positionalrelationship such that the coding sequence is expressed in an avianoviduct. The vector includes, but is not limited to, an avian leukosisvirus (ALV) retroviral vector, a murine leukemia virus (MLV) retroviralvector, and a lentivirus vector. The promoter is sufficient foreffecting expression of the coding sequence in the avian oviduct. Thecoding sequence codes for an exogenous protein which is deposited intothe egg white of a hard shell egg. As such, the coding sequence codesfor exogenous proteins such as transgenic poultry derived interferon-α2b (TPD IFN-α 2b) or transgenic poultry derived erythropoietin (TPDEPO). The vector used in the methods of the invention contains apromoter which is particularly suited for expression of exogenousproteins in avians and their eggs. As such, expression of the exogenouscoding sequence occurs in the oviduct and blood of the transgenic avianand in the egg white of its avian egg. The promoter includes, but is notlimited to, a cytomegalovirus (CMV) promoter, a MDOT promoter, arous-sarcoma virus (RSV) promoter, a murine leukemia virus (MLV)promoter, a mouse mammary tumor virus (MMTV) promoter, an ovalbuminpromoter, a lysozyme promoter, a conalbumin promoter, an ovomucoidpromoter, an ovomucin promoter, and an ovotransferrin promoter.Optionally, the promoter may be a segment of at least one promoterregion, such as a segment of the ovalbumin-, lysozyme-, conalbumin-,ovomucoid-, ovomucin-, and ovotransferrin promoter region.

One aspect of the invention involves truncating the ovalbumin promoterand/or condensing the critical regulatory elements of the ovalbuminpromoter so that it retains sequences required for expression in thetubular gland cells of the magnum of the oviduct, while being smallenough that it can be readily incorporated into vectors. For instance, asegment of the ovalbumin promoter region may be used. This segmentcomprises the 5′-flanking region of the ovalbumin gene. The total lengthof the ovalbumin promoter segment may be from about 0.88 kb to about 7.4kb in length, and is preferably from about 0.88 kb to about 1.4 kb inlength. The segment preferably includes both the steroid-dependentregulatory element and the negative regulatory element of the ovalbumingene. The segment optionally also includes residues from the5′untranslated region (5′UTR) of the ovalbumin gene. Alternatively, thepromoter may be a segment of the promoter region of the lysozyme-,conalbumin-, ovomucin-, ovomucoid- and ovotransferrin genes. An exampleof such a promoter is the synthetic MDOT promoter which is comprised ofelements from the ovomucoid (MD) and ovotransferrin (OT) promoter.

In another aspect of the invention, the vectors integrated into theavian genome contain constitutive promoters which are operably linked tothe exogenous coding sequence (e.g., cytomegalovirus (CMV) promoter,rous-sarcoma virus (RSV) promoter, and a murine leukemia virus (MLV)promoter. Alternatively, a non-constitutive promoter such as a mousemammary tumor virus (MMTV) promoter may be used.

Other aspects of the invention provide for transgenic avians which carrya transgene in the genetic material of their germ-line tissue. Morespecifically, the transgene includes an exogenous gene and a promoter inoperational and positional relationship to express the exogenous gene.The exogenous gene is expressed in the avian oviduct and in the blood ofthe transgenic avian. The exogenous gene codes for exogenous proteinssuch as TPD IFN-α 2b or TPD EPO. The exogenous protein is deposited intothe egg white of a hard shell egg.

Another aspect of the invention provides for an avian egg which containsprotein exogenous to the avian species. Use of the invention allows forexpression of exogenous proteins in oviduct cells with secretion of theproteins into the lumen of the oviduct magnum and deposition into theegg white of the avian egg. Proteins packaged into eggs may be presentin quantities of up to one gram or more per egg. The exogenous proteinincludes, but is not limited to, TPD IFN-α 2b and TPD EPO.

Still another aspect of the invention provides an isolatedpolynucleotide sequence comprising the optimized coding sequence ofhuman interferon-α 2b (IFN-α 2b), i.e., recombinant transgenic poultryderived interferon-α 2b coding sequence which codes for transgenicpoultry derived interferon-α 2b (TPD IFN-α 2b). The invention alsoencompasses an isolated protein comprising the polypeptide sequence ofTPD IFN-α 2b, wherein the protein is O-glycosylated at Thr-106 withN-Acetyl-Galactosamine, Galactose, N-Acetyl-Glucosamine, Sialic acid,and combinations thereof.

The invention further contemplates a pharmaceutical compositioncomprising the polypeptide sequence of TPD IFN-α 2b, wherein the proteinis O-glycosylated at Thr-106 with N-Acetyl-Galactosamine, Galactose,N-Acetyl-Glucosamine, Sialic acid, and combinations thereof.

Another aspect of the invention provides an isolated polynucleotidesequence comprising the optimized coding sequence of humanerythropoietin (EPO), i.e., recombinant transgenic poultry derivederythropoietin coding sequence which codes for transgenic poultryderived erythropoietin (TPD EPO).

Yet another aspect of the invention provides for a vector comprising afirst and second coding sequence and a promoter in operational andpositional relationship to the first and second coding sequence toexpress the first and second coding sequence in an avian oviduct. Thevector further includes an internal ribosome entry site (IRES) elementpositioned between the first and second coding sequence, wherein thefirst coding sequence codes for protein X and the second coding sequencecodes for protein Y, and wherein protein X and protein Y are depositedinto the egg white of a hard shell egg. For example, protein X may be alight chain (LC) of a monoclonal antibody and protein Y may be a heavychain (HC) of a monoclonal antibody. Alternatively, the protein encodedby the second coding sequence (e.g., enzyme) may be capable of providingpost-translational modification of the protein encoded by the firstcoding sequence. The vector optionally includes additional codingsequences and additional IRES elements, such that each coding sequencein the vector is separated from another coding sequence by an IRESelement.

The invention also contemplates methods of producing an avian egg whichcontains proteins such as monoclonal antibodies, enzymes, or otherproteins. Such a method includes providing a vector with a promoter,coding sequences, and at least one IRES element; creating transgeniccells or tissue by introducing the vector into avian embryonicblastodermal cells, wherein the vector sequence is randomly insertedinto the avian genome; and deriving a mature transgenic avian from thetransgenic cells or tissue. The transgenic avian so derived expressesthe coding sequences in its oviduct, and the resulting protein issecreted into the oviduct lumen, so that the protein is deposited intothe egg white of a hard shell egg.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate ovalbumin promoter expression vectorscomprising ovalbumin promoter segments and a coding sequence, gene X,which encodes an exogenous protein X. X represents any exogenous gene orexogenous protein of interest.

FIGS. 2A, 2B, 2C and 2D illustrate retroviral vectors of the inventioncomprising an ovalbumin promoter and a coding sequence, gene X, encodingan exogenous protein X. X represents any exogenous gene or exogenousprotein of interest.

FIG. 2E illustrates a method of amplifying an exogenous gene forinsertion into the vectors of 2A and 2B.

FIG. 2F illustrates a retroviral vector comprising an ovalbumin promotercontrolling expression of a coding sequence, gene X, and an internalribosome entry site (IRES) element enabling expression of a secondcoding sequence, gene Y. X and Y represent any gene of interest.

FIGS. 3A and 3B show schematic representations of the ALV-derivedvectors pNLB and pNLB-CMV-BL, respectively. Because NLB has not beensequenced in its entirety, measurements in bp (base pair) are estimatedfrom published data (Cosset et al., 1991; Thoraval et al., 1995) anddata discussed herein. The vectors are both shown as they would appearwhile integrated into the chicken genome.

FIGS. 4A and 4B show the amount of β-lactamase (lactamase) in the bloodserum of chimeric and transgenic chickens. In FIG. 4A the concentrationof bioactive lactamase in the serum of G0 chickens transduced with theNLB-CMV-BL transgene was measured at 8 month post-hatch. The generation,sex and wing band numbers are indicated. Lactamase serum concentrationswere measured for G1 transgenic chickens at 6 to 7 months post-hatch.Arrows indicate G1 chickens bred from rooster 2395. In FIG. 4B thelactamase serum concentration was measured for G1 and G2 transgenicchickens. Arrows indicate G2s bred from hen 5657 or rooster 4133.Samples from chickens 4133, 5308, and 5657 are the same as those in FIG.4A. Samples from G2 birds bred from 5657 were collected at 3 to 60 dayspost-hatch. Samples from G2 birds bred from 4133 were collected at 3month post-hatch.

FIG. 5 shows the pedigree of chickens bearing the transgenic lociharbored by hen 5657 (FIG. 5A) or rooster 4133 (FIG. 5B). 2395 was arooster that carried multiple transgenic loci. 2395 was bred to anon-transgenic hen, yielding 3 offspring each carrying the transgene ina unique position of the chicken genome. For simplicity, transgenicprogeny for which expression data were not shown as well asnon-transgenic progeny were omitted from the pedigree. Band numbers areindicated by the following symbols: ∘ hen; □ rooster; ● hen carrying theNLB-CMV-BL transgene; ▪ rooster carrying the NLB-CMV-BL transgene.

FIG. 6 shows β-lactamase (lactamase) in the egg white of hen 5657 andher offspring. In FIG. 6A egg white from hen 5657 and her transgenicoffspring were assayed for active lactamase. The control is fromuntreated hens and clutchmate is a non-transgenic G2 bred from hen 5657.Eggs were collected in March 2000. Arrows indicate G2s bred from hen5657. In FIG. 6B egg white samples from G2 transgenic hens carrying onecopy of the transgene (hemizygous) were compared with that of G3 hen6978 which harbored two copies (homozygous). Eggs were collected inFebruary 2001. The generation and wing band numbers are indicated to theleft.

FIG. 7 shows β-lactamase (lactamase) in the eggs of G2 and G3 hens bredfrom rooster 4133. In FIG. 7A egg whites from four representativehemizygous transgenic hens bred from rooster 4133 were assayed foractive lactamase. Eggs were collected in October 1999, March, 2000 andFebruary 2001 and a minimum of 4 eggs per hen were assayed one monthafter each set was collected. The control represents egg white fromuntreated hens. Band numbers are indicated to the left. The average ofthe 4 hens for each period is calculated. In FIG. 7B egg white fromhemizygous G2 transgenic hens were compared with that of hemizygous andhomozygous transgenic G3 hens. The eggs were collected in February 2001.The generation and transgene copy number are displayed in the data barfor each hen. The average concentration for hens carrying one or twocopies is at the bottom of the chart.

FIGS. 8A and 8B show the pNLB-CMV-IFN vector for expressing IFN-α 2b inchickens; and the pNLB-MDOT-EPO vector used for expressingerythropoietin (EPO) in chickens, respectively.

FIG. 9 depicts the novel glycosylation pattern of transgenic poultryderived interferon-α 2b (TPD IFN-α 2b), including all 6 bands.

FIG. 10 shows the comparison of human peripheral blood leukocyte derivedinterferon-α 2b (PBL IFN-α 2b or natural hIFN) and transgenic poultryderived interferon-α2b (TPD IFN-α 2b or egg white hIFN).

FIG. 11 depicts the synthetic amino acid sequence (residues 1-165) oftransgenic poultry derived interferon-α 2b (TPD IFN-α 2b) (SEQ ID NO:2).

FIG. 12A depicts the synthetic nucleic acid sequence (cDNA, residues1-579) of optimized human erythropoietin (EPO) i.e., recombinant TPD EPO(SEQ ID NO: 3). FIG. 12B depicts the synthetic amino acid sequence(residues 1-193) of transgenic poultry derived erythropoietin (TPD EPO)(SEQ ID NO: 4). (For natural human EPO see also NCBI Accession NumberNP_(—)000790).

FIG. 13 shows the synthetic MDOT promoter linked to the IFN-MM CDS. TheMDOT promoter contains elements from the chicken ovomucoid gene(ovomucoid promoter) ranging from −435 to −166 bp (see NCBI AccessionNumber J00894) and the chicken conalbumin gene (ovotransferrin promoter)ranging from −251 to +29 bp (see NCBI Accession Numbers Y00497, M11862and X01205).

FIG. 14 provides a summary of the major egg white proteins.

FIGS. 15A and 15D show the pCMV-LC-emcvIRES-HC vector, wherein the lightchain (LC) and heavy chain (HC) of a human monoclonal antibody wereexpressed from this single vector by placement of an IRES from theencephalomyocarditis virus (EMCV) in order to test for expression ofmonoclonal antibodies. In comparison, FIGS. 15B and 15C show theseparate vectors pCMV-HC and pCMV-LC, respectively, wherein thesevectors were also used to test for expression of monoclonal antibodies.

DETAILED DESCRIPTION OF THE INVENTION

-   a) Definitions and General Parameters

The following definitions are set forth to illustrate and define themeaning and scope of the various terms used to describe the inventionherein.

A “nucleic acid or polynucleotide sequence” includes, but is not limitedto, eukaryotic mRNA, cDNA, genomic DNA, and synthetic DNA and RNAsequences, comprising the natural nucleoside bases adenine, guanine,cytosine, thymidine, and uracil. The term also encompasses sequenceshaving one or more modified bases.

A “coding sequence” or “open reading frame” refers to a polynucleotideor nucleic acid sequence which can be transcribed and translated (in thecase of DNA) or translated (in the case of mRNA) into a polypeptide invitro or in vivo when placed under the control of appropriate regulatorysequences. The boundaries of the coding sequence are determined by atranslation start codon at the 5′ (amino) terminus and a translationstop codon at the 3′ (carboxy) terminus. A transcription terminationsequence will usually be located 3′ to the coding sequence. A codingsequence may be flanked on the 5′ and/or 3′ ends by untranslatedregions.

“Exon” refers to that part of a gene which, when transcribed into anuclear transcript, is “expressed” in the cytoplasmic mRNA after removalof the introns or intervening sequences by nuclear splicing.

Nucleic acid “control sequences” or “regulatory sequences” refer topromoter sequences, translational start and stop codons, ribosomebinding sites, polyadenylation signals, transcription terminationsequences, upstream regulatory domains, enhancers, and the like, asnecessary and sufficient for the transcription and translation of agiven coding sequence in a defined host cell. Examples of controlsequences suitable for eukaryotic cells are promoters, polyadenylationsignals, and enhancers. All of these control sequences need not bepresent in a recombinant vector so long as those necessary andsufficient for the transcription and translation of the desired gene arepresent.

“Operably or operatively linked” refers to the configuration of thecoding and control sequences so as to perform the desired function.Thus, control sequences operably linked to a coding sequence are capableof effecting the expression of the coding sequence. A coding sequence isoperably linked to or under the control of transcriptional regulatoryregions in a cell when DNA polymerase will bind the promoter sequenceand transcribe the coding sequence into mRNA that can be translated intothe encoded protein. The control sequences need not be contiguous withthe coding sequence, so long as they function to direct the expressionthereof. Thus, for example, intervening untranslated yet transcribedsequences can be present between a promoter sequence and the codingsequence and the promoter sequence can still be considered “operablylinked” to the coding sequence.

The terms “heterologous” and “exogenous” as they relate to nucleic acidsequences such as coding sequences and control sequences, denotesequences that are not normally associated with a region of arecombinant construct or with a particular chromosomal locus, and/or arenot normally associated with a particular cell. Thus, an “exogenous”region of a nucleic acid construct is an identifiable segment of nucleicacid within or attached to another nucleic acid molecule that is notfound in association with the other molecule in nature. For example, anexogenous region of a construct could include a coding sequence flankedby sequences not found in association with the coding sequence innature. Another example of an exogenous coding sequence is a constructwhere the coding sequence itself is not found in nature (e.g., syntheticsequences having codons different from the native gene). Similarly, ahost cell transformed with a construct which is not normally present inthe host cell would be considered exogenous for purposes of thisinvention.

“Exogenous protein” as used herein refers to a protein not naturallypresent in a particular tissue or cell, a protein that is the expressionproduct of an exogenous expression construct or transgene, or a proteinnot naturally present in a given quantity in a particular tissue orcell.

“Endogenous gene” refers to a naturally occurring gene or fragmentthereof normally associated with a particular cell.

The expression products described herein may consist of proteinaceousmaterial having a defined chemical structure. However, the precisestructure depends on a number of factors, particularly chemicalmodifications common to proteins. For example, since all proteinscontain ionizable amino and carboxyl groups, the protein may be obtainedin acidic or basic salt form, or in neutral form. The primary amino acidsequence may be derivatized using sugar molecules (glycosylation) or byother chemical derivatizations involving covalent or ionic attachmentwith, for example, lipids, phosphate, acetyl groups and the like, oftenoccurring through association with saccharides. These modifications mayoccur in vitro, or in vivo, the latter being performed by a host cellthrough posttranslational processing systems. Such modifications mayincrease or decrease the biological activity of the molecule, and suchchemically modified molecules are also intended to come within the scopeof the invention.

Alternative methods of cloning, amplification, expression, andpurification will be apparent to the skilled artisan. Representativemethods are disclosed in Sambrook, Fritsch, and Maniatis, MolecularCloning, a Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory(1989).

“Vector” means a polynucleotide comprised of single strand, doublestrand, circular, or supercoiled DNA or RNA. A typical vector may becomprised of the following elements operatively linked at appropriatedistances for allowing functional gene expression: replication origin,promoter, enhancer, 5′ mRNA leader sequence, ribosomal binding site,nucleic acid cassette, termination and polyadenylation sites, andselectable marker sequences. One or more of these elements may beomitted in specific applications. The nucleic acid cassette can includea restriction site for insertion of the nucleic acid sequence to beexpressed. In a functional vector the nucleic acid cassette contains thenucleic acid sequence to be expressed including translation initiationand termination sites. An intron optionally may be included in theconstruct, preferably ≧100 bp 5′ to the coding sequence. A vector isconstructed so that the particular coding sequence is located in thevector with the appropriate regulatory sequences, the positioning andorientation of the coding sequence with respect to the control sequencesbeing such that the coding sequence is transcribed under the “control”of the control or regulatory sequences. Modification of the sequencesencoding the particular protein of interest may be desirable to achievethis end. For example, in some cases it may be necessary to modify thesequence so that it may be attached to the control sequences with theappropriate orientation; or to maintain the reading frame. The controlsequences and other regulatory sequences may be ligated to the codingsequence prior to insertion into a vector. Alternatively, the codingsequence can be cloned directly into an expression vector which alreadycontains the control sequences and an appropriate restriction site whichis in reading frame with and under regulatory control of the controlsequences.

A “promoter” is a site on the DNA to which RNA polymerase binds toinitiate transcription of a gene. In some embodiments the promoter willbe modified by the addition or deletion of sequences, or replaced withalternative sequences, including natural and synthetic sequences as wellas sequences which may be a combination of synthetic and naturalsequences. Many eukaryotic promoters contain two types of recognitionsequences: the TATA box and the upstream promoter elements. The former,located upstream of the transcription initiation site, is involved indirecting RNA polymerase to initiate transcription at the correct site,while the latter appears to determine the rate of transcription and isupstream of the TATA box. Enhancer elements can also stimulatetranscription from linked promoters, but many function exclusively in aparticular cell type. Many enhancer/promoter elements derived fromviruses, e.g., the SV40 promoter, the cytomegalovirus (CMV) promoter,the rous-sarcoma virus (RSV) promoter, and the murine leukemia virus(MLV) promoter are all active in a wide array of cell types, and aretermed “constitutive” or “ubiquitous”. Alternatively, non-constitutivepromoters such as the mouse mammary tumor virus (MMTV) promoter may alsobe used in the instant invention. The nucleic acid sequence inserted inthe cloning site may have any open reading frame encoding a polypeptideof interest, with the proviso that where the coding sequence encodes apolypeptide of interest, it should lack cryptic splice sites which canblock production of appropriate mRNA molecules and/or produce aberrantlyspliced or abnormal mRNA molecules.

A “marker gene” is a gene which encodes a protein that allows foridentification and isolation of correctly transfected cells. Suitablemarker sequences include, but are not limited to green, yellow, and bluefluorescent protein genes (GFP, YFP, and BFP, respectively). Othersuitable markers include thymidine kinase (tk), dihydrofolate reductase(DHFR), and aminoglycoside phosphotransferase (APH) genes. The latterimparts resistance to the aminoglycoside antibiotics, such as kanamycin,neomycin, and geneticin. These, and other marker genes such as thoseencoding chloramphenicol acetyltransferase (CAT), β-lactamase,β-galactosidase (β-gal), may be incorporated into the primary nucleicacid cassette along with the gene expressing the desired protein, or theselection markers may be contained on separate vectors andcotransfected.

A “reporter gene” is a marker gene that “reports” its activity in a cellby the presence of the protein that it encodes.

A “retroviral particle”, “transducing particle”, or “transductionparticle” refers to a replication-defective or replication-competentvirus capable of transducing non-viral DNA or RNA into a cell.

The terms “transformation”, “transduction” and “transfection” all denotethe introduction of a polynucleotide into an avian blastodermal cell.

“Magnum” is that part of the oviduct between the infundibulum and theisthmus containing tubular gland cells that synthesize and secrete theegg white proteins of the egg.

A “MDOT promoter”, as used herein, is a synthetic promoter which isactive in the tubular gland cells of the magnum of the oviduct amongstother tissues. MDOT is comprised of elements from the ovomucoid (MD) andovotransferrin (OT) promoters (FIG. 13).

The term “optimized” is used in the context of “optimized codingsequence”, wherein the most frequently used codons for each particularamino acid found in the egg white proteins ovalbumin, lysozyme,ovomucoid, and ovotransferrin are used in the design of the optimizedhuman interferon-α 2b (IFN-α 2b) polynucleotide sequence that isinserted into vectors of the instant invention. More specifically, theDNA sequence for optimized human IFN-α 2b is based on the hen oviductoptimized codon usage and is created using the BACKTRANSLATE program ofthe Wisconsin Package, Version 9.1 (Genetics Computer Group Inc.,Madison, Wis.) with a codon usage table compiled from the chicken(Gallus gallus) ovalbumin, lysozyme, ovomucoid, and ovotransferrinproteins. For example, the percent usage for the four codons of theamino acid alanine in the four egg white proteins is 34% for GCU, 31%for GCC, 26% for GCA, and 8% for GCG. Therefore, GCU is used as thecodon for the majority of alanines in the optimized human IFN-α 2bcoding sequence. The vectors containing the gene for optimized humanIFN-α 2b are used to create transgenic avians that express transgenicpoultry derived IFN-α 2b (TPD IFN-α 2b) in their tissues and eggs.Similarly, the above method is employed for the design of the optimizedhuman erythropoietin (EPO) polynucleotide sequence in order to createtransgenic avians that express transgenic poultry derived erythropoietin(TPD EPO) in their tissues and eggs.

-   b) Novel Vectors and Transgensis of Blastodermal Cells

By the methods of the present invention, transgenes can be introducedinto avian embryonic blastodermal cells, to produce a transgenic chickenor transgenic turkey, or other avian species, that carries the transgenein the genetic material of its germ-line tissue. The blastodermal cellsare typically stage VII-XII cells, or the equivalent thereof, andpreferably are near stage X. The cells useful in the present inventioninclude embryonic germ (EG) cells, embryonic stem (ES) cells &primordial germ cells (PGCs). The embryonic blastodermal cells may beisolated freshly, maintained in culture, or reside within an embryo.

The vectors useful in carrying out the methods of the present inventionare described herein. These vectors may be used for stable introductionof an exogenous coding sequence into the genome of an avian.Alternatively, the vectors may be used to produce exogenous proteins inspecific tissues of an avian, and in the oviduct in particular. Thevectors may also be used in methods to produce avian eggs which containexogenous protein. In a preferred embodiment, the vector is retroviraland the coding sequence and the promoter are both positioned between the5′ and 3′ LTRs of the retroviral vector. In another preferredembodiment, the retroviral vector is derived from the avian leukosisvirus (ALV), murine leukemia virus (MLV), or lentivirus. In anotherpreferred embodiment, the vector includes a signal peptide codingsequence which is operably linked to the coding sequence, so that upontranslation in a cell, the signal peptide will direct secretion of theexogenous protein expressed by the vector into the egg white of a hardshell egg. In yet another preferred embodiment, the vector furtherincludes a marker gene, wherein said marker gene is operably linked tothe promoter.

In some cases, introduction of a vector of the present invention intothe embryonic blastodermal cells is performed with embryonicblastodermal cells that are either freshly isolated or in culture. Thetransgenic cells are then typically injected into the subgerminal cavitybeneath a recipient blastoderm in an egg. In some cases, however, thevector is delivered directly to the cells of a blastodermal embryo.

In one embodiment of the invention, vectors used for transfectingblastodermal cells and generating random, stable integration into theavian genome contain a coding sequence and a promoter in operational andpositional relationship to express the coding sequence in the tubulargland cell of the magnum of the avian oviduct, wherein the codingsequence codes for an exogenous protein which is deposited in the eggwhite of a hard shell egg. The promoter may optionally be a segment ofthe ovalbumin promoter region which is sufficiently large to directexpression of the coding sequence in the tubular gland cells. Theinvention involves truncating the ovalbumin promoter and/or condensingthe critical regulatory elements of the ovalbumin promoter so that itretains sequences required for expression in the tubular gland cells ofthe magnum of the oviduct, while being small enough that it can bereadily incorporated into vectors. In a preferred embodiment, a segmentof the ovalbumin promoter region may be used. This segment comprises the5′-flanking region of the ovalbumin gene. The total length of theovalbumin promoter segment may be from about 0.88 kb to about 7.4 kb inlength, and is preferably from about 0.88 kb to about 1.4 kb in length.The segment preferably includes both the steroid-dependent regulatoryelement and the negative regulatory element of the ovalbumin gene. Thesegment optionally also includes residues from the 5′ untranslatedregion (5′ UTR) of the ovalbumin gene. Hence, the promoter may bederived from the promoter regions of the ovalbumin-, lysozyme-,conalbumin-, ovomucoid-, ovotransferrin- or ovomucin genes (FIG. 14). Anexample of such a promoter is the synthetic MDOT promoter which iscomprised of elements from the ovomucoid and ovotransferrin promoter(FIG. 13). The promoter may also be a promoter that is largely, but notentirely, specific to the magnum, such as the lysozyme promoter. Thepromoter may also be a mouse mammary tumor virus (MMTV) promoter.Alternatively, the promoter may be a constitutive promoter (e.g., acytomegalovirus (CMV) promoter, a rous-sarcoma virus (RSV) promoter, amurine leukemia virus (MLV) promoter, etc.). In a preferred embodimentof the invention, the promoter is a cytomegalovirus (CMV) promoter, aMDOT promoter, a rous-sarcoma virus (RSV) promoter, a murine leukemiavirus (MLV) promoter, a mouse mammary tumor virus (MMTV) promoter, anovalbumin promoter, a lysozyme promoter, a conalbumin promoter, anovomucoid promoter, an ovomucin promoter, and an ovotransferrinpromoter. Optionally, the promoter may be at least one segment of apromoter region, such as a segment of the ovalbumin-, lysozyme-,conalbumin-, ovomucoid-, ovomucin-, and ovotransferrin promoter region.In a particularly preferred embodiment, the promoter is a CMV promoter.

FIGS. 1A and 1B illustrate examples of ovalbumin promoter expressionvectors. Gene X is a coding sequence which encodes an exogenous protein.Bent arrows indicate the transcriptional start sites. In one example,the vector contains 1.4 kb of the 5′ flanking region of the ovalbumingene (FIG. 1A). The sequence of the “−1.4 kb promoter” of FIG. 1Acorresponds to the sequence starting from approximately 1.4 kb upstream(1.4 kb) of the ovalbumin transcription start site and extendingapproximately 9 residues into the 5′ untranslated region of theovalbumin gene. The approximately 1.4 kb-long segment harbors twocritical regulatory elements, the steroid-dependent regulatory element(SDRE) and the negative regulatory element (NRE). The NRE is so namedbecause it contains several negative regulatory elements which block thegene's expression in the absence of hormones (e.g., estrogen). A shorter0.88 kb segment also contains both elements. In another example, thevector contains approximately 7.4 kb of the 5′ flanking region of theovalbumin gene and harbors two additional elements (HS-III and HS-IV),one of which is known to contain a functional region enabling inductionof the gene by estrogen (FIG. 1B). A shorter 6 kb segment also containsall four elements and could optionally be used in the present invention.

Each vector used for random integration according to the presentinvention preferably comprises at least one 1.2 kb element from thechicken β-globin locus which insulates the gene within from bothactivation and inactivation at the site of insertion into the genome. Ina preferred embodiment, two insulator elements are added to one end ofthe ovalbumin gene construct. In the β-globin locus, the insulatorelements serve to prevent the distal locus control region (LCR) fromactivating genes upstream from the globin gene domain, and have beenshown to overcome position effects in transgenic flies, indicating thatthey can protect against both positive and negative effects at theinsertion site. The insulator element(s) are only needed at either the5′ or 3′ end of the gene because the transgenes are integrated inmultiple, tandem copies effectively creating a series of genes flankedby the insulator of the neighboring transgene. In another embodiment,the insulator element is not linked to the vector but is cotransfectedwith the vector. In this case, the vector and the element are joined intandem in the cell by the process of random integration into the genome.

Each vector may optionally also comprise a marker gene to allowidentification and enrichment of cell clones which have stablyintegrated the expression vector. The expression of the marker gene isdriven by a ubiquitous promoter that drives high levels of expression ina variety of cell types. In a preferred embodiment of the invention, themarker gene is human interferon driven by a lysozyme promoter. Inanother embodiment the green fluorescent protein (GFP) reporter gene(Zolotukhin et al., J. Virol 70:4646-4654 (1995)) is driven by theXenopus elongation factor 1-α (ef-1α) promoter (Johnson and Krieg, Gene147:223-26 (1994)). The Xenopus ef-1α promoter is a strong promoterexpressed in a variety of cell types. The GFP contains mutations thatenhance its fluorescence and is humanized, or modified such that thecodons match the codon usage profile of human genes. Since avian codonusage is virtually the same as human codon usage, the humanized form ofthe gene is also highly expressed in avian blastodermal cells. Inalternative embodiments, the marker gene is operably linked to one ofthe ubiquitous promoters of HSV tk, CMV, β-actin, or RSV.

While human and avian codon usage is well matched, where a nonvertebrategene is used as the coding sequence in the transgene, the nonvertebrategene sequence may be modified to change the appropriate codons such thatcodon usage is similar to that of humans and avians.

Transfection of the blastodermal cells may be mediated by any number ofmethods known to those of ordinary skill in the art. The introduction ofthe vector to the cell may be aided by first mixing the nucleic acidwith polylysine or cationic lipids which help facilitate passage acrossthe cell membrane. However, introduction of the vector into a cell ispreferably achieved through the use of a delivery vehicle such as aliposome or a virus. Viruses which may be used to introduce the vectorsof the present invention into a blastodermal cell include, but are notlimited to, retroviruses, adenoviruses, adeno-associated viruses, herpessimplex viruses, and vaccinia viruses.

In one method of transfecting blastodermal cells, a packagedretroviral-based vector is used to deliver the vector into embryonicblastodermal cells so that the vector is integrated into the aviangenome.

As an alternative to delivering retroviral transduction particles to theembryonic blastodermal cells in an embryo, helper cells which producethe retrovirus can be delivered to the blastoderm.

A preferred retrovirus for randomly introducing a transgene into theavian genome is the replication-deficient avian leucosis virus (ALV),the replication-deficient murine leukemia virus (MLV), or thelentivirus. In order to produce an appropriate retroviral vector, a pNLBvector is modified by inserting a region of the ovalbumin promoter andone or more exogenous genes between the 5′ and 3′ long terminal repeats(LTRs) of the retrovirus genome. Any coding sequence placed downstreamof the ovalbumin promoter will be expressed in the tubular gland cellsof the oviduct magnum because the ovalbumin promoter drives theexpression of the ovalbumin protein and is active in the oviduct tubulargland cells. While a 7.4 kb ovalbumin promoter has been found to producethe most active construct when assayed in cultured oviduct tubular glandcells, the ovalbumin promoter is preferably shortened for use in theretroviral vector. In a preferred embodiment, the retroviral vectorcomprises a 1.4 kb segment of the ovalbumin promoter; a 0.88 kb segmentwould also suffice.

Any of the vectors of the present invention may also optionally includea coding sequence encoding a signal peptide that will direct secretionof the protein expressed by the vector's coding sequence from thetubular gland cells of the oviduct. This aspect of the inventioneffectively broadens the spectrum of exogenous proteins that may bedeposited in avian eggs using the methods of the invention. Where anexogenous protein would not otherwise be secreted, the vector bearingthe coding sequence is modified to comprise a DNA sequence comprisingabout 60 bp encoding a signal peptide from the lysozyme gene. The DNAsequence encoding the signal peptide is inserted in the vector such thatit is located at the N-terminus of the protein encoded by the cDNA.

FIGS. 2A-2D illustrate examples of suitable retroviral vectorconstructs. The vector construct is inserted into the avian genome with5′ and 3′ flanking LTRs. Neo is the neomycin phosphotransferase gene.Bent arrows indicate transcription start sites. FIGS. 2A and 2Billustrate LTR and oviduct transcripts with a sequence encoding thelysozyme signal peptide (LSP), whereas FIGS. 2C and 2D illustratetranscripts without such a sequence. There are two parts to theretroviral vector strategy. Any protein that contains a eukaryoticsignal peptide may be cloned into the vectors depicted in FIGS. 2B and2D. Any protein that is not ordinarily secreted may be cloned into thevectors illustrated in FIGS. 2A and 2B to enable its secretion from thetubular gland cells.

FIG. 2E illustrates the strategy for cloning an exogenous gene into alysozyme signal peptide vector. The polymerase chain reaction is used toamplify a copy of a coding sequence, gene X, using a pair ofoligonucleotide primers containing restriction enzyme sites that enablethe insertion of the amplified gene into the plasmid after digestionwith the two enzymes. The 5′ and 3′ oligonucleotides contain the Bsu36Iand Xba1 restriction sites, respectively.

Another aspect of the invention involves the use of internal ribosomeentry site (IRES) elements in any of the vectors of the presentinvention to allow the translation of two or more proteins from a di- orpolycistronic mRNA (Example 15). The IRES units are fused to 5′ ends ofone or more additional coding sequences which are then inserted into thevectors at the end of the original coding sequence, so that the codingsequences are separated from one another by an IRES (FIGS. 2F, 15A and15D). Pursuant to this aspect of the invention, post-translationalmodification of the product is facilitated because one coding sequencemay encode an enzyme capable of modifying the other coding sequenceproduct. For example, the first coding sequence may encode collagenwhich would be hydroxylated and made active by the enzyme encoded by thesecond coding sequence. In the retroviral vector example of FIG. 2F, aninternal ribosome entry site (IRES) element is positioned between twoexogenous coding sequences (gene X and gene Y). The IRES allows bothprotein X and protein Y to be translated from the same transcriptdirected by an ovalbumin promoter. Bent arrows indicate transcriptionstart sites. The expression of the protein encoded by gene X is expectedto be highest in tubular gland cells, where it is specifically expressedbut not secreted. The protein encoded by gene Y is also expressedspecifically in tubular gland cells but because it is efficientlysecreted, protein Y is packaged into the eggs. In the retroviral vectorexample of FIGS. 15A and 15D, the light chain (LC) and heavy chain (HC)of a human monoclonal antibody are expressed from a single vector,pCMV-LC-emcvIRES-HC, by placement of an IRES from theencephalomyocarditis virus (EMCV). Transcription is driven by a CMVpromoter. (See also Murakami et al. (1997) “High-level expression ofexogenous genes by replication-competent retrovirus vectors with aninternal ribosomal entry site” Gene 202:23-29; Chen et al. (1999)“Production and design of more effective avian replication-incompetentretroviral vectors” Dev. Biol. 214:370-384; Noel et al. (2000)“Sustained systemic delivery of monoclonal antibodies by geneticallymodified skin fibroblasts” J. Invest. Dermatol. 115:740-745.)

In another aspect of the invention, the coding sequences of vectors usedin any of the methods of the present invention are provided with a 3′untranslated region (3′ UTR) to confer stability to the RNA produced.When a 3′ UTR is added to a retroviral vector, the orientation of thefused ovalbumin promoter, gene X and the 3′ UTR must be reversed in theconstruct, so that the addition of the 3′ UTR will not interfere withtranscription of the full-length genomic RNA. In a preferred embodiment,the 3′ UTR may be that of the ovalbumin or lysozyme genes, or any 3′ UTRthat is functional in a magnum cell, i.e., the SV40 late region.

In an alternative embodiment of the invention, a constitutive promoter(e.g., CMV) is used to express the coding sequence of a transgene in themagnum of an avian. In this case, expression is not limited to themagnum; expression also occurs in other tissues within the avian (e.g.,blood). The use of such a transgene, which includes a constitutivepromoter and a coding sequence, is particularly suitable for effectingthe expression of a protein in the oviduct and the subsequent secretionof the protein into the egg white (see FIG. 8A for an example of a CMVdriven construct, such as the pNLB-CMV-IFN vector for expressing IFN-α2b in chickens).

FIG. 3A shows a schematic of the replication-deficient avian leukosisvirus (ALV)-based vector PNLB, a vector which is suitable for use inthis embodiment of the invention. In the pNLB vector, most of the ALVgenome is replaced by the neomycin resistance gene (Neo) and the lacZgene, which encodes b-galactosidase. FIG. 3B shows the vectorpNLB-CMV-BL, in which lacZ has been replaced by the CMV promoter and theβ-lactamase coding sequence (β-La or BL). Construction of the vector isreported in the specific examples (Example 1, vide infra). β-lactamaseis expressed from the CMV promoter and utilizes a polyadenylation signal(pA) in the 3′ long terminal repeat (LTR). The β-Lactamase protein has anatural signal peptide; thus, it is found in blood and in egg white.

Avian embryos are transduced with the pNLB-CMV-BL vector (Example 2,vide infra). The egg whites of eggs from the resulting stably transducedhens contain up to 60 micrograms (μg) of secreted, active β-lactamaseper egg (Examples 2 and 3, vide infra).

FIGS. 8A and 8B illustrates the pNLB-CMV-IFN vector used for expressinginterferon-α 2b (IFN-α 2b) and the pNLB-MDOT-EPO vector used forexpressing erythropoietin (EPO), respectively. Both exogenous proteins(EPO, IFN) are expressed in avians, preferably chicken and turkey.

The pNLB-MDOT-EPO vector is created by substituting an EPO encodingsequence for the BL encoding sequence (Example 10, vide infra). In oneembodiment, a synthetic promoter called MDOT is employed to driveexpression of EPO. MDOT contains elements from both the ovomucoid andovotransferrin promoter. The DNA sequence for human EPO is based on henoviduct optimized codon usage as created using the BACKTRANSLATE programof the Wisconsin Package, version 9.1 (Genetics Computer Group, Inc.,Madison, Wis.) with a codon usage table compiled from the chicken(Gallus gallus) ovalbumin, lysozyme, ovomucoid, and ovotransferrinproteins. The EPO DNA sequence is synthesized and cloned into the vectorand the resulting plasmid is pNLB-MDOT-EPO (a.k.a. pAVIJCR-A145.27.2.2).In one embodiment, transducing particles (i.e., transduction particles)are produced for the vector, and these transducing particles are titeredto determine the appropriate concentration that can be used to injectembryos. Eggs are then injected with transducing particles after whichthey hatch about 21 days later. The exogenous protein levels such as theEPO levels can then be measured by an ELISA assay from serum samplescollected from chicks one week after hatch. Male birds are selected forbreeding, wherein birds are screened for G₀ roosters which contain theEPO transgene in their sperm. Preferably, roosters with the highestlevels of the transgene in their sperm samples are bred to nontransgenichens by artificial insemination. Blood DNA samples are screened for thepresence of the transgene. A number of chicks are usually found to betransgenic (G₁ avians). Chick serum is tested for the presence of humanEPO (e.g, ELISA assay). The egg white in eggs from G₁ hens is alsotested for the presence of human EPO. The EPO (i.e., derived from theoptimized coding sequence of human EPO) present in eggs of the instantinvention is biologically active (Example 11).

Similarly, the pNLB-CMV-IFN vector (FIG. 8A) is created by substitutingan IFN encoding sequence for the BL encoding sequence (Example 12, videinfra). In one embodiment, a constitutive cytomegalovirus (CMV) promoteris employed to drive expression of IFN. More specifically, the IFNcoding sequence is controlled by the cytomegalovirus (CMV) immediateearly promoter/enhancer and SV40 polyA site. FIG. 8A illustratespNLB-CMV-IFN used for expressing IFN in avians, preferably chicken andturkey. An optimized coding sequence is created for human IFN-α 2b,wherein the most frequently used codons for each particular amino acidfound in the egg white proteins ovalbumin, lysozyme, ovomucoid, andovotransferrin are used in the design of the human IFN-α 2b sequencethat is inserted into vectors of the instant invention. Morespecifically, the DNA sequence for the optimized human IFN-α 2b (FIG.11A) is based on the hen oviduct optimized codon usage and is createdusing the BACKTRANSLATE program (supra) with a codon usage tablecompiled from the chicken (Gallus gallus) ovalbumin, lysozyme,ovomucoid, and ovotransferrin proteins. For example, the percent usagefor the four codons of the amino acid alanine in the four egg whiteproteins is 34% for GCU, 31% for GCC, 26% for GCA, and 8% for GCG.Therefore, GCU is used as the codon for the majority of alanines in theoptimized human IFN-α 2b sequence. The vectors containing the gene forthe optimized human IFN-α 2b sequence are used to create transgenicavians that express TPD IFN-α 2b in their tissues and eggs.

Transducing particles (i.e., transduction particles) are produced forthe vector and titered to determine the appropriate concentration thatcan be used to inject embryos (Example 2, vide infra). Thus, chimericavians are produced (see also Example 13, vide infra). Avian eggs arewindowed according to the Speksnijder procedure (U.S. Pat. No.5,897,998), and eggs are injected with transducing particles. Eggs hatchabout 21 days after injection. hIFN levels are measured (e.g., ELISAassay) from serum samples collected from chicks one week after hatch. Aswith EPO (supra), male birds are selected for breeding. In order toscreen for G₀ roosters which contain the IFN transgene in their sperm,DNA is extracted from rooster sperm samples. The G₀ roosters with thehighest levels of the transgene in their sperm samples are bred tonontransgenic hens by artificial insemination. Blood DNA samples arescreened for the presence of the transgene. The serum of transgenicroosters is tested for the presence of hIFN (e.g., ELISA assay). If theexogenous protein is confirmed the sperm of the transgenic roosters isused for artificial insemination of nontransgenic hens. A certainpercent of the offspring will then contain the transgene (e.g., morethan 50%). When IFN (i.e., derived from the optimized coding sequence ofhuman IFN) is present in eggs of the instant invention, the IFN may betested for biological activity. As with EPO, such eggs usually containbiologically active IFN, such as TPD IFN-α 2b (FIG. 11B).

-   c) Production of Transgenic Avians and Exogenous Proteins in Eggs

The methods of the invention which provide for the production ofexogenous protein in the avian oviduct and the production of eggs whichcontain exogenous protein involve an additional step subsequent toproviding a suitable vector and introducing the vector into embryonicblastodermal cells so that the vector is integrated into the aviangenome. The subsequent step involves deriving a mature transgenic avianfrom the transgenic blastodermal cells produced in the previous steps.Deriving a mature transgenic avian from the blastodermal cellsoptionally involves transferring the transgenic blastodermal cells to anembryo and allowing that embryo to develop fully, so that the cellsbecome incorporated into the avian as the embryo is allowed to develop.The resulting chick is then grown to maturity. In a preferredembodiment, the cells of a blastodermal embryo are transfected ortransduced with the vector directly within the embryo (Example 2). TheResulting Embryo is Allowed to Develop and the Chick Allowed to Mature.

In either case, the transgenic avian so produced from the transgenicblastodermal cells is known as a founder. Some founders will carry thetransgene in the tubular gland cells in the magnum of their oviducts.These avians will express the exogenous protein encoded by the transgenein their oviducts. The exogenous protein may also be expressed in othertissues (e.g., blood) in addition to the oviduct. If the exogenousprotein contains the appropriate signal sequence(s), it will be secretedinto the lumen of the oviduct and into the egg white of the egg. Somefounders are germ-line founders (Examples 8 and 9). A germ-line founderis a founder that carries the transgene in genetic material of itsgerm-line tissue, and may also carry the transgene in oviduct magnumtubular gland cells that express the exogenous protein. Therefore, inaccordance with the invention, the transgenic avian will have tubulargland cells expressing the exogenous protein, and the offspring of thetransgenic avian will also have oviduct magnum tubular gland cells thatexpress the exogenous protein. Alternatively, the offspring express aphenotype determined by expression of the exogenous gene in specifictissue(s) of the avian (Example 6, Table 2). In a preferred embodimentof the invention, the transgenic avian is a chicken or a turkey.

The invention can be used to express, in large yields and at low cost, awide range of desired proteins including those used as human and animalpharmaceuticals, diagnostics, and livestock feed additives. Proteinssuch as interferon (IFN), erythropoietin (EPO), human growth hormone,lysozyme, and β-casein are examples of proteins which are desirablyexpressed in the oviduct and deposited in eggs according to theinvention (Examples 2, 3, and 5). Other possible proteins to be producedinclude, but are not limited to, albumin, α-1 antitrypsin, antithrombinIII, collagen, factors VIII, IX, X (and the like), fibrinogen,hyaluronic acid, insulin, lactoferrin, protein C, granulocytecolony-stimulating factor (G-CSF), granulocyte macrophagecolony-stimulating factor (GM-CSF), tissue-type plasminogen activator(tPA), feed additive enzymes, somatotropin, and chymotrypsin.Genetically engineered antibodies, such as immunotoxins which bind tosurface antigens on human tumor cells and destroy them, can also beexpressed for use as pharmaceuticals or diagnostics.

-   d) Transgenic Poultry Derived Interferon-α 2b (TPD IFN-α 2b)

The instant invention encompasses a transgenic poultry derivedinterferon-α 2b (TPD IFN-α 2b) derived from avians. TPD IFN-α 2bexhibits a new glycosylation pattern and contains two new glyco forms(bands 4 and 5 are α-Gal extended disaccharides; see FIG. 9) notnormally seen in human peripheral blood leukocyte derived interferon-α2b (PBL IFN-α 2b). TPD IFN-α 2b also contains O-linked carbohydratestructures that are similar to human PBL IFN-α 2b and is moreefficiently produced in chickens then the human form.

The instant invention contemplates an isolated polynucleotide comprisingthe optimized polynucleotide sequence of human IFN-α 2b, i.e.,recombinant transgenic poultry derived interferon-α 2b (TPD IFN-α 2b)coding sequence (SEQ ID NO: 1). The coding sequence for optimized humanIFN-α 2b includes 498 nucleic acids and 165 amino acids (see SEQ ID NO:1 and FIG. 11A). Similarly, the coding sequence for natural human IFN-α2b includes 498 nucleic acids (NCBI Accession Number AF405539 andGI:15487989) and 165 amino acids (NCBI Accession Number AAL01040 andGI:15487990). The most frequently used codons for each particular aminoacid found in the egg white proteins ovalbumin, lysozyme, ovomucoid, andovotransferrin are used in the design of the optimized human IFN-α 2bcoding sequence which is inserted into vectors of the instant invention.More specifically, the DNA sequence for the optimized human IFN-α 2b isbased on the hen oviduct optimized codon usage and is created using theBACKTRANSLATE program of the Wisconsin Package, Version 9.1 (GeneticsComputer Group Inc., Madison, Wis.) with a codon usage table compiledfrom the chicken (Gallus gallus) ovalbumin, lysozyme, ovomucoid, andovotransferrin proteins. For example, the percent usage for the fourcodons of the amino acid alanine in the four egg white proteins is 34%for GCU, 31% for GCC, 26% for GCA, and 8% for GCG. Therefore, GCU isused as the codon for the majority of alanines in the optimized humanIFN-α 2b coding sequence. The vectors containing the gene for optimizedhuman IFN-α 2b are used to create transgenic avians that express TPDIFN-α 2b in their tissues and eggs.

As discussed in Example 13 (vide infra), TPD IFN-α 2b is produced inchicken. However, TPD IFN-α 2b may also be produced in turkey and otheravian species. In a preferred embodiment of the invention, TPD IFN-α 2bis expressed in chicken and turkey and their hard shell eggs. Acarbohydrate analysis (Example 14, vide infra), including amonosaccharide analysis and FACE analysis, reveals the sugar make-up ornovel glycosylation pattern of the protein. As such, TPD IFN-α 2b showsthe following monosaccharide residues: N-Acetyl-Galactosamine (NAcGal),Galactose (Gal), N-Acetyl-Glucosamine (NAcGlu), and Sialic acid (SA).However, there is no N-linked glycosylation in TPD IFN-α 2b. Instead,TPD IFN-α 2b is O-glycosylated at Thr-106. This type of glycosylation issimilar to human IFN-α 2, wherein the Thr residue at position 106 isunique to IFN-α 2. Similar to natural IFN-α, TPD IFN-α 2b does not havemannose residues. A FACE analysis reveals 6 bands (FIG. 9) thatrepresent various sugar residues, wherein bands 1, 2 and 3 areun-sialyated, mono-sialyated, and di-sialyated, respectively (FIG. 10).The sialic acid (SA) linkage is alpha 2-3 to Galactose (Gal) and alpha2-6 to N-Acetyl-Galactosamine (NAcGal). Band 6 represents anun-sialyated tetrasaccharide. Bands 4 and 5 are alpha-Galactose(alpha-Gal) extended disaccharides that are not seen in human PBL IFN-α2b or natural human IFN (natural hIFN). FIG. 10 shows the comparison ofTPD IFN-α 2b (egg white HIFN) and human PBL IFN-α 2b (natural hIFN).Minor bands are present between bands 3 and 4 and between bands 4 and 5in TPD IFN-α 2b (vide infra).

The instant invention contemplates an isolated polypeptide sequence (SEQID NO: 2) of TPD IFN-α 2b (see also FIG. 11B) and a pharmaceuticalcomposition thereof, wherein the protein is O-glycosylated at Thr-106with specific residues. These residues are as follows:

In a preferred embodiment of the instant invention, the percentages areas follows:

Minor bands are present between bands 3 and 4 and between bands 4 and 5which account for about 17% in TPD IFN-α 2b.

e) EXAMPLES

The following specific examples are intended to illustrate the inventionand should not be construed as limiting the scope of the claims.

Example 1 Vector Construction

The lacZ gene of pNLB, a replication-deficient avian leukosis virus(ALV)-based vector (Cosset et al., 1991), was replaced with anexpression cassette consisting of a cytomegalovirus (CMV) promoter andthe reporter gene, β-lactamase. The pNLB and pNLB-CMV-BL vectorconstructs are diagrammed in FIGS. 3A and 3B, respectively.

To efficiently replace the lacZ gene of pNLB with a transgene, anintermediate adaptor plasmid was first created, pNLB-Adapter.pNLB-Adapter was created by inserting the chewed back ApaI/ApaI fragmentof pNLB (Cosset et al., J. Virol. 65:3388-94 (1991)) (in pNLB, the 5′ApaI resides 289 bp upstream of lacZ and the 3′ApaI resides 3′ of the 3′LTR and Gag segments) into the chewed-back KpnI/SacI sites ofpBluescriptKS(−). The filled-in MluI/XbaI fragment of pCMV-BL (Moore etal., Anal. Biochem. 247:203-9 (1997)) was inserted into the chewed-backKpnI/NdeI sites of pNLB-Adapter, replacing lacZ with the CMV promoterand the BL gene (in pNLB, KpnI resides 67 bp upstream of lacZ and NdeIresides 100 bp upstream of the lacZ stop codon), thereby creatingpNLB-Adapter-CMV-BL. To create pNLB-CMV-BL, the HindIII/BlpI insert ofpNLB (containing lacZ) was replaced with the HindIII/BlpI insert ofpNLB-Adapter-CMV-BL. This two step cloning was necessary because directligation of blunt-ended fragments into the HindIII/BlpI sites of pNLByielded mostly rearranged subclones, for unknown reasons.

Example 2 Creation of the NLB-CMV-BL Founder Flock

Sentas and Isoldes were cultured in F10 (Gibco), 5% newborn calf serum(Gibco), 1% chicken serum (Gibco), 50 μg/ml phleomycin (CaylaLaboratories) and 50 μg/ml hygromycin (Sigma). Transduction particleswere produced as described in Cosset et al., 1993, herein incorporatedby reference, with the following exceptions. Two days after transfectionof the retroviral vector pNLB-CMV-BL (from Example 1, above) into 9×10⁵Sentas, virus was harvested in fresh media for 6-16 hours and filtered.All of the media was used to transduce 3×10⁶ Isoldes in 3 100 mm plateswith polybrene added to a final concentration of 4 μg/ml. The followingday the media was replaced with media containing 50 μg/ml phleomycin, 50μg/ml hygromycin and 200 μg/ml G418 (Sigma). After 10-12 days, singleG418^(r) colonies were isolated and transferred to 24-well plates. After7-10 days, titers from each colony were determined by transduction ofSentas followed by G418 selection. Typically 2 out of 60 colonies gavetiters at 1-3×10⁵. Those colonies were expanded and virus concentratedto 2-7×10⁶ as described in Allioli et al., Dev. Biol. 165:30-7 (1994),herein incorporated by reference. The integrity of the CMV-BL expressioncassette was confirmed by assaying for β-lactamase in the media of cellstransduced with NLB-CMV-BL transduction particles.

The transduction vector, NLB-CMV-BL, was injected into the subgerminalcavity of 546 unincubated SPF White Leghorn embryos, of which 126 chickshatched and were assayed for secretion of β-lactamase (lactamase) intoblood. In order to measure the concentration of active lactamase inunknown samples, a kinetic colorimetric assay was employed in whichPADAC, a purple substrate, is converted to a yellow compoundspecifically by lactamase. Lactamase activity was quantitated bymonitoring the decrease in OD_(570 nm) during a standard reaction timeand compared to a standard curve with varying levels of purifiedlactamase (referred to as the “lactamase assay”). The presence orabsence of lactamase in a sample could also be determined by visuallyscoring for the conversion of purple to yellow in a test sampleovernight or for several days (the “overnight lactamase assay”). Thelatter method was suitable for detection of very low levels of lactamaseor for screening a large number of samples. At one to four weeks of age,chick serum samples were tested for the presence of lactamase.Twenty-seven chicks had very low levels of lactamase in their serum thatwas detectable only after the overnight lactamase assay and, as thesebirds matured, lactamase was no longer detectable. As shown in Table 1below and FIG. 4A, 9 additional birds (3 males and 6 females) had serumlevels of lactamase that ranged from 11.9 to 173.4 ng/ml at six to sevenmonths post-hatch.

TABLE 1 Expression of β-Lactamase in NLB-CMV-BL-Transduced ChickensAverage ng/ml of β-Lactamase Egg White: Egg White: Serum: 8 8 Month 14Month Sex Band No. Month Birds Hens³ Hens³ NA¹ Controls²  0.0 ± 7.4  0.0± 13.6  0.0 ± 8.0 Female 1522 36.7 ± 1.6  56.3 ± 17.8  47.9 ± 14.3Female 1549 11.9 ± 1.3 187.0 ± 32.4 157.0 ± 32.2 Female 1581 31.5 ± 4.8243.8 ± 35.7 321.7 ± 68.8 Female 1587 33.9 ± 1.4 222.6 ± 27.7 291.0 ±27.0 Female 1790 31.0 ± 0.5 136.6 ± 20.2 136.3 ± 11.0 Female 1793 122.8± 3.6  250.0 ± 37.0 232.5 ± 28.6 Male 2395 16.0 ± 2.3 NA NA Male 2421165.5 ± 5.0  NA NA Male 2428 173.4 ± 5.9  NA NA ¹NA: not applicable.²Controls were obtained from untreated hens. ³Represents the average of5 to 20 eggs.

Example 3 β-Lactamase Expression in the Egg White of G0 Hens

Fifty-seven pullets transduced with NLB-CMV-BL retroviral vector wereraised to sexual maturity and egg white from each hen was tested foractive β-lactamase (lactamase) at 8 months of age. Of the 57 birds, sixhad significant levels of lactamase that ranged from 56.3 to 250.0 ng/ml(Table 1, supra). No other hens in this group had detectable levels oflactamase in their egg white, even after incubation of PADAC with thesample for several days. Lactamase was not detectable in egg white from24 hens that were mock injected and in 42 hens that were transduced witha NLB vector that did not carry the lactamase transgene. Stablelactamase expression was still detectable in the egg white of the sixexpressing hens six months following the initial assays (Table 1,supra).

Lactamase was detected in the egg white of all six hens by a westernblot assay with an anti-β-lactamase antibody. The egg white lactamasewas the same size as the bacterially produced, purified lactamase thatwas used as a standard. The amount detected in egg white by Westernanalysis was consistent with that determined by the enzymatic assay,indicating that a significant proportion of the egg white lactamase wasbiologically active. Hen-produced lactamase in egg white stored at 4° C.lost no activity and showed no change in molecular weight even afterseveral months of storage. This observation allowed storage oflactamase-containing eggs for extended periods prior to analysis.

Example 4 Germline Transmission and Serum Expression of the β-LactamaseTransgene in G1 and G2 Transgenic Chickens

DNA was extracted from sperm collected from 56 G0 roosters and three ofthe 56 birds that harbored significant levels of the transgene in theirsperm DNA as determined by quantitative PCR were selected for breeding.These roosters were the same three that had the highest levels ofβ-lactamase (lactamase) in their blood (roosters 2395, 2421 and 2428).Rooster 2395 gave rise to three G1 transgenic offspring (out of 422progeny) whereas the other two yielded no transgenic offspring out of630 total progeny. Southern analysis of blood DNA from each of the threeG1 transgenic chickens confirmed that the transgenes were intact andthat they were integrated at unique random loci. The serum of the G1transgenic chicks, 5308, 5657 and 4133, at 6 to 11 weeks post-hatchcontained 0.03, 2.0 and 6.0 μg/ml of lactamase, respectively. The levelsof lactamase dropped to levels of 0.03, 1.1 and 5.0 μg/ml when thechickens were assayed again at 6 to 7 months of age (FIG. 4A).

Hen 5657 and rooster 4133 were bred to non-transgenic chickens to obtainoffspring hemizygous for the transgene. The pedigrees of transgenicchickens bred from rooster 4133 or hen 5657 and the subsequentgenerations are shown in FIG. 5. Transgenic rooster 5308 was also bredbut this bird's progeny exhibited lactamase concentrations that wereeither very low or not detectable in serum and egg white. Activelactamase concentrations in the serum of randomly selected G2 transgenicchicks were measured at 3 to 90 days post-hatch. Of the five G2transgenics bred from hen 5657, all had active lactamase atconcentrations of 1.9 to 2.3 μg/ml (compared to the parental expressionof 1.1 μg/ml, FIG. 4B). All of the samples were collected during thesame period of time, thus, the lactamase concentrations in the serum ofthe offspring were expected to be higher than that of the parent sincethe concentration in hen 5657 had dropped proportionately as shematured. Similarly, the five randomly selected transgenic chicks bredfrom rooster 4133 all had serum lactamase concentrations that weresimilar but higher than that of their parent (FIG. 4B).

Example 5 β-Lactamase Expression in the Egg White of Transgenic Hens

Eggs from G1 hen 5657 contained 130 ng of active β-lactamase (lactamase)per ml of egg white (FIG. 6A). Lactamase concentrations were higher inthe first few eggs laid and then reached a plateau that was stable forat least nine months. Eggs from transgenic hens bred from hen 5657 and anon-transgenic rooster had lactamase concentrations that were similar totheir parent (FIG. 6A). Hen 6978 was bred from G2 hen 8617 and siblingG2 rooster 8839 and was homozygous for the transgene as determined byquantitative PCR and Southern analysis. As expected, the concentrationof lactamase in the eggs of bird 6978 was nearly two-fold higher thanher hemizygous parent (FIG. 6B). No other G3 hens bred from hen 5657were analyzed because hen 6978 was the only female in her clutch. It isimportant to note that the eggs from hens 8867, 8868 and 8869 werecollected eleven months apart and had similar concentrations oflactamase (FIGS. 6A and 6B), again indicating that the expression levelsin the egg white were consistent throughout the lay period.

Rooster 4133 was bred to non-transgenic hens to obtain hemizygous G2hens. Of the 15 transgenic hens analyzed, all had lactamase in the eggwhite at concentrations ranging from 0.47 to 1.34 μg/ml. Fourrepresentative hens are shown in FIG. 7A. When assayed 6 months later,the average expression level had dropped from approximately 1.0 μg/ml to0.8 μg/ml (FIG. 7A). Expression levels were high in the initial eggs andleveled out over several months. After that, the concentrations oflactamase in the eggs remained constant.

G2 hen 8150 and sibling G2 rooster 8191 were crossed to yield hemizygousand homozygous G3 hens. All transgenic G3 hens expressed lactamase inthe white of their eggs at concentrations ranging from 0.52 to 1.65μg/ml (FIG. 7B). The average expression for the G3 hens that werehomozygous was 47% higher than those G2 hens and G3 hens that werehemizygous. The amount of lactamase in the eggs from G2 and G3 hens bredfrom rooster 4133 and his offspring varied significantly (FIGS. 7A and7B), although the levels in the eggs from any given hen in that groupwere relatively constant. The average expression of lactamase wasexpected to double for the homozygous genotype. Western blot analysisconfirmed that the transgene was faithfully producing intact lactamasein the eggs of G2 transgenics. The lactamase level detected on a Westernblot also correlated closely with that determined by the enzyme activityassay, indicating that a significant portion of the egg white lactamasewas bioactive. Thus, retroviral vectors were successfully employed toimplement stable and reliable expression of a transgene in chickens.

Deposition of lactamase in the yolk was detectable but lower than thatof egg white. Seven G2 or G3 hens of rooster 4133's lineage wereanalyzed and the concentration in the yolk ranged from 107 to 375 ng/mlor about 20% the concentration in the egg white. There was nocorrelation between the yolk and egg white lactamase levels of a givenhen (Harvey et al., “Expression of exogenous protein in egg white oftransgenic chickens” (April 2002) Nat. Biotechnol. 20:396-399).

Example 6 Production of Founder Males

For NLB-CMV-BL transduction, freshly laid fertilized White Leghorn eggswere used. Seven to ten microliters of concentrated particles wereinjected into the subgerminal cavity of windowed eggs and chicks hatchedafter sealing the window. 546 eggs were injected. Blood DNA wasextracted and analyzed for the presence of the transgene using aprobe-primer set designed to detect the neo^(r) gene via the Taqmanassay. As can be seen in Table 2 below, approximately 25% of all chickshad detectable levels of transgene in their blood DNA.

TABLE 2 Summary of Transgenesis with the NLB-CMV-BL Vectors TransgeneNLB-CMV-BL Number of injections 546 Number of birds hatched (%) 126(23.1%) Number of chicks with transgene in  36 (28.6%) their blood DNA(%) Number of males  56 Number of males with transgene in  3 (5.4%)their sperm DNA (%)

TABLE 2 Summary of Transgenesis with the NLB-CMV-BL Vectors ContinuedProduction Number of chicks bred from G0 males 1026 of G1 flock Numberof G1 transgenics 3 Rate of germline transmission 0.29% Production of G2Number of chicks bred from G1 120 flock transgenics Number of G2transgenics 61 Rate of germline transmission 50.8% Number of males thattransmitted 1 (1.8%) transgene to progeny (%)

Example 7 Germline Transmission of the Transgene

Taqman detection of the neo^(r) gene in sperm DNA was used to identifycandidate G0 males for breeding. Three G0 males were identified, whereineach had the NLB-CMV-BL transgene in their sperm DNA at levels that wereabove background. All G0 males positive for the transgene in their spermwere bred to non-transgenic hens to identify fully transgenic G1offspring.

For NLB-CMV-BL 1026 chicks were bred, respectively, and three G1 chicksobtained for each transgene (Table 2, supra). All G1 progeny came fromthe male with the highest level of transgene in his sperm DNA, eventhough an equivalent number of chicks were bred from each male.

Example 8 Southern Analysis of G1s and G2s

In order to confirm integration and integrity of the inserted vectorsequences, Southern blot analysis was performed on DNA from G1 and G2transgenics. Blood DNA was digested with HindIII and hybridized to aneo^(r) probe to detect junction fragments created by the internalHindIII site found in the NLB-CMV-BL vector (FIG. 3B) and genomic sitesflanking the site of integration. Each of the 3 G1 birds carryingNLB-CMV-BL had a junction fragment of unique size, indicating that thetransgene had integrated into three different genomic sites. G1s werebred to non-transgenic hens to obtain hemizygous G2s. As can be seen inTable 2 (supra), 50.8% of offspring from G1 roosters harboringNLB-CMV-BL were transgenic as expected for Mendelian segregation of asingle integrated transgene. Southern analysis of HindIII-digested DNAfrom G2 offspring detected junction fragments similar in size to thoseoriginating from their transgenic parents, indicating that the transgenewas transmitted intact.

Example 9 Screening for G3 Progeny Homozygous for the Transgene

In order to obtain transgenic chickens homozygous for the transgene, G2hemizygous birds bearing NLB-CMV-BL integrated at the same site (e.g.,progeny of the same G1 male) were crossbred. Two groups were bred: thefirst was a hen and rooster arising from the G1 4133 male and the secondfrom the G1 5657 hen. The Taqman assay was used to quantitatively detectthe neo^(r) transgene in G3 progeny using a standard curve. The standardcurve was constructed using known amounts of genomic DNA from the G1transgenic 4133 male hemizygous for the transgene as determined bySouthern analysis. The standard curve ranged from 10³ to 1.6×10⁴ totalcopies of the transgene or 0.2 to 3.1 transgene copies per diploidgenome. Because reaction components were not limited during theexponential phase, amplification was very efficient and gavereproducible values for a given copy number. There was a reproducible,one-cycle difference between each standard curve differing 2-fold incopy number.

In order to determine the number of transgene alleles in the G3offspring, DNAs were amplified and compared to the standards. DNA fromnon-transgenics did not amplify. Birds homozygous for the transgenicallele gave rise to plots initiating the amplification one cycle earlierthan those hemizygous for the allele. The sequence detection program wasable to calculate the number of alleles in an unknown DNA sample basedon the standard curve and the cycle threshold (Ct) at which a sample'samplification plot exhibited a significant rise. The data are shown inTable 3 below.

In order to confirm Taqman copy number analysis, DNA of selected birdswas analyzed by Southern blotting using PstI-digested DNA and a probecomplementary to the neo^(r) gene to detect a 0.9 kb fragment. Detectionof a small fragment was chosen since transfer of smaller DNAs from gelto membrane is more quantitative. The signal intensity of the 0.9 kbband corresponded well to the copy number of G3 transgenic birds asdetermined by the Taqman assay. The copy numbers of an additionaleighteen G3 transgenic birds analyzed by Southern blotting were alsoconsistent with that determined by Taqman. A total of 33 progeny wereanalyzed for the 4133 lineage, of which 9 (27.3%) were non-transgenic,16 (48.5%) were hemizygous and 8 (24.2%) were homozygous. A total of 10progeny were analyzed for the 5657 lineage, of which 5 (50.0%) werenon-transgenic, 1 (10.0%) was hemizygous and 4 (40.0%) were homozygous.The observed ratio of non-transgenics, hemizygotes and homozygotes forthe 4133 lineage G3 progeny was not statistically different from theexpected 1:2:1 ratio as determined by the χ² test (P≦0.05). Progeny ofthe 5657 lineage did not have the expected distribution but this couldhave been due to the low number of progeny tested (Harvey et al.,“Consistent production of transgenic chickens using replicationdeficient retroviral vectors and high-throughput screening procedures”(February 2002) Poultry Science 81:202-212).

TABLE 3 Determination of Transgene Copy Number in G3 Offspring Bred fromG2 Transgenics Band No. Copies per (Std. No. or Mean Total StandardDiploid G1 Parent NTC¹) Ct² Copy Number Deviation Genome³ NA⁴ 4133 27.33,975 145.7 1 4133 6792 40.0 0 0.0 0 5657 6977 25.9 10,510 587.0 2 56576978 25.8 10,401 505.1 2 4133 7020 26.7 6,064 443.1 1 4133 7021 26.85,239 133.8 1 4133 7022 26.1 9,096 352.3 2 4133 7023 26.8 5,424 55.7 14133 7024 26.9 4,820 110.1 1 5657 7110 26.4 8,092 1037.5 2 5657 711130.4 403 46.3 0 5657 7112 33.2 60 6.1 0 4133 7142 26.5 6,023 367.6 14133 7143 25.9 9,474 569.8 2 4133 7144 25.7 12,420 807.7 2 4133 733827.2 4,246 201.7 1 5657 7407 37.7 1 1.0 0 NA (std1) 29.1 1,000 0.0 0.2NA (std2) 28.1 2,000 0.0 0.4 NA (std3) 27.1 4,000 0.0 0.8 NA (std4) 26.28,000 0.0 1.6 NA (std5) 25.3 16,000 0.0 3.1 NA (NTC) 39.8 −1 0.0 0.0¹Std. No.: standard number; NTC: no template control. ²Ct: cyclethreshold; cycle at which a sample's fluorescence exhibited asignificant increase above background. ³Copies per diploid genome weredetermined by dividing the mean by 5100 and rounding to the nearestfirst decimal place. ⁴NA: not applicable.

Example 10 Vector Construction for pNLB-MDOT-EPO Vector

Following the teachings of Example 1 (Vector Construction) of thespecification, an pNLB-MDOT-EPO vector was created, substituting an EPOencoding sequence for the BL encoding sequence (FIG. 8B). Instead ofusing the CMV promoter MDOT was used (FIG. 13). MDOT is a syntheticpromoter which contains elements from both the ovomucoid (MD) andovotransferrin (OT) promoter. (pNLB-MDOT-EPO vector, a.k.a.pAVIJCR-A145.27.2.2).

The DNA sequence for human EPO based on hen oviduct optimized codonusage was created using the BACKTRANSLATE program of the WisconsinPackage, version 9.1 (Genetics Computer Group, Inc., Madison, Wis.) witha codon usage table compiled from the chicken (Gallus gallus) ovalbumin,lysozyme, ovomucoid, and ovotransferrin proteins. The DNA sequence wassynthesized and cloned into the 3′ overhang T's of pCRII-TOPO(Invitrogen) by Integrated DNA Technologies, Coralville, Iowa, on acontractual basis. The EPO coding sequence was then removed from pEpoMMwith Hind III and Fse I, purified from a 0.8% agarose-TAE Gel, andligated to Hind III and Fse I digested, alkaline phosphatase-treatedpCMV-IFNMM. The resulting plasmid was pAVIJCR-A137.43.2.2 whichcontained the EPO coding sequence controlled by the cytomegalovirusimmediate early promoter/enhancer and SV40 polyA site. The plasmidpAVIJCR-A137.43.2.2 was digested with Nco I and Fse I and theappropriate fragment ligated to an Nco I and Fse I-digested fragment ofpMDOTIFN to obtain pAVIJCR-A137.87.2.1 which contained EPO driven by theMDOT promoter. In order to clone the EPO coding sequence controlled bythe MDOT promoter into the NLB retroviral plasmid, the plasmidspALVMDOTIFN and pAVIJCR-A137.87.2.1 were digested with Kpn I and Fse I.Appropriate DNA fragments were purified on a 0.8% agarose-TAE gel, thenligated and transformed into DH5α cells. The resulting plasmid waspNLB-MDOT-EPO (a.k.a. pAVIJCR-A145.27.2.2).

Example 11 Production of Transgenic Chickens and Fully Transgenic G1Chickens Expressing EPO

Production of NLB-MDOT-EPO transduction particles were performed asdescribed for NLB-CMV-BL (see Example 2). Approximately 300 WhiteLeghorn eggs were windowed according to the Speksnijder procedure (U.S.Pat. No. 5,897,998), then injected with ˜7×10⁴ transducing particles peregg. Eggs hatched 21 days after injection, and human EPO levels weremeasured by EPO ELISA from serum samples collected from chicks one weekafter hatch.

In order to screen for G₀ roosters which contained the EPO transgene intheir sperm, DNA was extracted from rooster sperm samples by Chelex-100extraction (Walsh et al., 1991). DNA samples were then subjected toTaqman™ analysis on a 7700 Sequence Detector (Perkin Elmer) using the“neo for-1” (5′-TGGATTGCACGCAGGTTCT-3′ SEQ ID NO: 3) and “neo rev-1”(5′-GTGCCCAGTCATAGCCGAAT-3′ SEQ ID NO: 4) primers and FAM labeledNEO-PROBE1 (5′-CCTCTCCACCCAAGCGGCCG-3′ SEQ ID NO: 5) to detect thetransgene. Eight G₀ roosters with the highest levels of the transgene intheir sperm samples were bred to nontransgenic SPAFAS (White Leghorn)hens by artificial insemination. Blood DNA samples were screened for thepresence of the transgene by Taqman™ analysis as described above.

Out of 1,054 offspring, 16 chicks were found to be transgenic (G1avians). Chick serum was tested for the presence of human EPO by EPOELISA, and EPO was present at ˜70 nanogram/ml (ng/ml). Egg white in eggsfrom G1 hens was also tested for the presence of human EPO by EPO ELISAand found to contain human EPO at ˜70 ng/ml. The EPO present in eggs(i.e., derived from the optimized coding sequence of human EPO) wasfound to be biologically active when tested on a human EPO responsivecell line (HCD57 murine erythroid cells) in a cell culture assay.

Example 12 Vector Construction for pNLB-CMV-IFN

Following the teachings of Example 1, a pNLB-CMV-IFN vector was created(FIG. 8A), substituting an IFN encoding sequence for the BL encodingsequence of Example 1.

An optimized coding sequence was created, wherein the most frequentlyused codons for each particular amino acid found in the egg whiteproteins ovalbumin, lysozyme, ovomucoid, and ovotransferrin were used inthe design of the optimized human IFN-α 2b coding sequence that wasinserted into vectors of the instant invention. More specifically, theDNA sequence for optimized human IFN-α 2b is based on the hen oviductoptimized codon usage and was created using the BACKTRANSLATE program ofthe Wisconsin Package, Version 9.1 (Genetics Computer Group Inc.,Madison, Wis.) with a codon usage table compiled from the chicken(Gallus gallus) ovalbumin, lysozyme, ovomucoid, and ovotransferrinproteins. For example, the percent usage for the four codons of theamino acid alanine in the four egg white proteins is 34% for GCU, 31%for GCC, 26% for GCA, and 8% for GCG. Therefore, GCU was used as thecodon for the majority of alanines in the optimized human IFN-α 2bcoding sequence. The vectors containing the gene for optimized humanIFN-α 2b were used to create transgenic avians that express transgenicpoultry derived interferon-α 2b (TPD IFN-α 2b) in their tissues andeggs.

The template and primer oligonucleotides listed in Table 4 below wereamplified by PCR with Pfu polymerase (Stratagene, La Jolla, Calif.)using 20 cycles of 94° C. for 1 min.; 50° C. for 30 sec.; and 72° C. for1 min. and 10 sec. PCR products were purified from a 12%polyacrylamide-TBE gel by the “crush and soak” method (Maniatis et al.1982), then combined as templates in an amplification reaction usingonly IFN-1 and IFN-8 as primers (see Table 4). The resulting PCR productwas digested with Hind III and Xba I and gel purified from a 2%agarose-TAE gel, then ligated into Hind III and Xba I digested, alkalinephosphatase-treated pBluescript KS (Stratagene), resulting in theplasmid pBluKSP-IFNMagMax. Both strands were sequenced by cyclesequencing on an ABI PRISM 377 DNA Sequencer (Perkin-Elmer, Foster City,Calif.) using universal T7 or T3 primers. Mutations in pBluKSP-IFNderived from the original oligonucleotide templates were corrected bysite-directed mutagenesis with the Transformer Site-Directed MutagenesisKit (Clontech, Palo Alto, Calif.). The IFN coding sequence was thenremoved from the corrected pBluKSP-IFN with Hind III and Xba 1, purifiedfrom a 0.8% agarose-TAE Gel, and ligated to Hind III and Xba I digested,alkaline phosphatase-treated pCMV-BetaLa-3B-dH. The resulting plasmidwas pCMV-IFN which contained an IFN coding sequence controlled by thecytomegalovirus immediate early promoter/enhancer and SV40 polyA site.In order to clone the IFN coding sequence controlled by the CMVpromoter/enhancer into the NLB retroviral plasmid, pCMV-IFN was firstdigested with ClaI and XbaI, then both ends were filled in with Klenowfragment of DNA polymerase (New England BioLabs, Beverly, Mass.).pNLB-adapter was digested with NdeI and KpnI, and both ends were madeblunt by T4 DNA polymerase (New England BioLabs). Appropriate DNAfragments were purified on a 0.8% agarose-TAE gel, then ligated andtransformed into DH5β cells. The resulting plasmid waspNLB-adapter-CMV-IFN. This plasmid was then digested with MluI andpartially digested with BlpI and the appropriate fragment was gelpurified. pNLB-CMV-EGFP was digested with MluI and BlpI, thenalkaline-phosphatase treated and gel purified. The MluI/BlpI partialfragment of pNLB-adapter-CMV-IFN was ligated to the large fragmentderived from the MluI/BlpI digest of pNLB-CMV-EGFP creatingpNLB-CMV-IFN.

TABLE 4 Oligonucleotides used for IFN Gene Synthesis Sequence of PrimerSequence of Primer Sequence of Template Template 1 Primer 1 2 Primer 2IFN-A 5′ATGGCTTTGACCTTTGCCTTA IFN-1 5′CCCAAGCTTTCACCA IFN-25′CTGTGGGTCTGAGG CTGGTGGCTCTCCTGGTGCTGA TGGCTTTGACCTTTGC CAGAT3′GCTGCAAGAGCAGCTGCTCTGT CTT3′ (SEQ ID NO:8) GGGCTGCGATCTGCCTCA3′ (SEQ IDNO:7) (SEQ ID NO:6) IFN-B 5′GACCCACAGCCTGGGCAGCA IFN-2b5′ATCTGCCTCAGACCC IFN-3b 5′AACTCCTCTTGAGG GGAGGACCCTGATGCTGCTGGC ACAG3′AAAGCCAAAATC3′ TCAGATGAGGAGAATCAGCCTG (SEQ ID NO:10) (SEQ ID NO:11)TTTAGCTGCCTGAAGGATAGGC ACGATTTTGGCTTT3′ (SEQ ID NO:9) IFN-C5′CTCAAGAGGAGTTTGGCAAC IFN-3c 5′GATTTTGGCTTTCCT IFN-4 5′ATCTGCTGGATCATCAGTTTCAGAAGGCTGAGACCA CAAGAGGAGTT3′ CTCGTGC3′ TCCCTGTGCTGCACGAGATG3′(SEQ ID NO:13) (SEQ ID NO:14) (SEQ ID NO:12) IFN-D5′ATCCAGCAGATCTTTAACCTG IFN-4b 5′GCACGAGATGATCC IFN-5 5′ATCGTTCAGCTGCTTTTAGCACCAAGGATAGCAGCG AGCAGAT3′ GGTACA3′ CTGCTTGGGATGAGACCCTGCT (SEQ IDNO:16) (SEQ ID NO:17) GGATAAGTTTTACACCGAGCTG TACCAGCA3′ (SEQ ID NO:15)IFN-E 5′GCTGAACGATCTGGAGGCTT IFN-5b 5′TGTACCAGCAGCTGA IFN-65′CCTCACAGCCAGGA GCGTGATCCAGGGCGTGGGCGT ACGAT 3′ TGCTAT3′GACCGAGACCCCTCTGATGAA (SEQ ID NO:19) (SEQ ID NO:20) GGAGGATAGCATCCT3′(SEQ ID NO:18) IFN-F 5′GGCTGTGAGGAAGTACTTTCA IFN-6b 5′ATAGCATCCTGGCTGIFN-7 5′ATGATCTCAGCCCT GAGGATCACCCTGTACCTGAAG TGAGG 3′ CACGAC3′GAGAAGAAGTACAGCCCTTGC (SEQ ID NO:22) (SEQ ID NO:23)GCTTGGGAAGTCGTGAGGG3′ (SEQ ID NO:21) IFN-G 5′CTGAGATCATGAGGAGCTTTAIFN-7b 5′GTCGTGAGGGCTGA IFN-8 5′TGCTCTAGACTTTT GCCTGAGCACCAACCTGCAAGGATCAT 3′ TACTCCTTAGACCTC AGAGCTTGAGGTCTAAGGAGTA (SEQ ID NO:25)AAGCTCT3′ A3′ (SEQ ID NO:26) (SEQ ID NO:24)

Example 13 Production of Transgenic Chickens and Fully Transgenic G1Chickens Expressing IFN

Transduction particles of pNLB-CMV-IFN were produced following theprocedures of Example 2. Approximately 300 White Leghorn (strain Line 0)eggs were windowed according to the Speksnijder procedure (U.S. Pat. No.5,897,998), then injected with ˜7×10⁴ transducing particles per egg.Eggs hatched 21 days after injection, and human IFN levels were measuredby IFN ELISA from serum samples collected from chicks one week afterhatch.

In order to screen for G₀ roosters which contained the IFN transgene intheir sperm, DNA was extracted from rooster sperm samples by Chelex-100extraction (Walsh et al., 1991). DNA samples were then subjected toTaqman™ analysis on a 7700 Sequence Detector (Perkin Elmer) using the“neo for-1” (5′-TGGATTGCACGCAGGTTCT-3′ SEQ ID NO: 3) arid “neo rev-1”(5′-GTGCCCAGTCATAGCCGAAT-3′ SEQ ID NO: 4) primers and FAM labeledNEO-PROBE1 (5′-CCTCTCCACCCAAGCGGCCG-3′ SEQ ID NO: 5) to detect thetransgene. Three G₀ roosters with the highest levels of the transgene intheir sperm samples were bred to nontransgenic SPAFAS (White Leghorn)hens by artificial insemination.

Blood DNA samples were screened for the presence of the transgene byTaqman™ analysis as described above. Out of 1,597 offspring, one roosterwas found to be transgenic (a.k.a. “Alphie”). Alphie's serum was testedfor the presence of hIFN by hIFN ELISA, and hIFN was present at 200ng/ml.

Alphie's sperm was used for artificial insemination of nontransgenicSPAFAS (White Leghorn) hens. 106 out of 202 (˜52%) offspring containedthe transgene as detected by Taqman™ analysis. These breeding resultsfollowed a Mendelian inheritance pattern and indicated that Alphie istransgenic.

Example 14 Carbohydrate Analysis of Transgenic Poultry DerivedInterferon-α 2b (TPD IFN-α 2b)

Experimental evidence revealed a new glycosylation pattern ininterferon-α 2b derived from avians (i.e., TPD IFN-α 2b). TPD IFN-α 2bwas found to contain two new glyco forms (bands 4 and 5 are α-Galextended disaccharides; see FIG. 9) not normally seen in humanperipheral blood leukocyte derived interferon-α 2b (PBL IFN-α 2b) ornatural human interferon-α 2b (natural HIFN). TPD IFN-α 2b was alsofound to contain O-linked carbohydrate structures that are similar tohuman PBL IFN-α 2b and was more efficiently produced in chickens thenthe human form.

The coding sequence for human IFN-α 2b was optimized (Example 12, supra)resulting in a recombinant IFN-α 2b coding sequence. TPD IFN-α 2b wasthen produced in chickens (Example 13, supra). A carbohydrate analysis,including a monosaccharide analysis and FACE analysis, revealed thesugar make-up or novel glycosylation pattern of the protein. As such,TPD IFN-α 2b showed the following monosaccharide residues:N-Acetyl-Galactosamine (NAcGal), Galactose (Gal), N-Acetyl-Glucosamine(NAcGlu), and Sialic acid (SA). No N-linked glycosylation was found inTPD IFN-α 2b. Instead, TPD IFN-α 2b was found to be O-glycosylated atThr-106. This type of glycosylation is similar to human IFN-α 2, whereinthe Thr residue at position 106 is unique to IFN-α 2. In addition, TPDIFN-α 2b was found to have no mannose residues. A FACE analysis revealed6 bands (FIG. 9) that represent various sugar residues, wherein bands 1,2 and 3 are un-sialyated, mono-sialyated, and di-sialyated, respectively(FIG. 10). The sialic acid (SA) linkage is alpha 2-3 to Galactose (Gal)and alpha 2-6 to N-Acetyl-Galactosamine (NAcGal). Band 6 represents anun-sialyated tetrasaccharide. Bands 4 and 5 were found to bealpha-Galactose (alpha-Gal) extended disaccharides that are not seen inhuman PBL IFN-α 2b. FIG. 10 shows the comparison of TPD IFN-α 2b (eggwhite hIFN) and human PBL IFN-α 2b (natural hIFN). Minor bands werepresent between bands 3 and 4 and between bands 4 and 5 in TPD IFN-α 2b(vide infra).

The protein was found to be O-glycosylated at Thr-106 with specificresidues, such as:

The percentages were as follows:

Minor bands were present between bands 3 and 4 and between bands 4 and 5which account for about 17% in TPD IFN-α 2b.

Example 15 Expression of MAbs From Plasmid Transfection and RetroviralTransduction Using the EMCV IRES in Avian Cells

The light chain (LC) and heavy chain (HC) of a human monoclonal antibodywere expressed from a single vector, pCMV-LC-emcvlRES-HC, by placementof an IRES from the encephalomyocarditis virus (EMCV) (see also Jang etal. (1988) “A segment of the 5′ nontranslated region ofencephalomyocarditis virus RNA directs internal entry of ribosomesduring in vitro translation” J. Virol. 62:2636-2643) between the LC andHC coding sequences. Transcription was driven by the CMV promoter.

In order to test expression of monoclonal antibodies from two separatevectors, the LC or HC linked to the CMV promoter were cotransfected intoLMH/2a cells, an estrogen-responsive, chicken hepatocyte cell line (seealso Binder et al. (1990) “Expression of endogenous and transfectedapolipoprotein II and vitellogenin II genes in an estrogen responsivechicken liver cell line” Mol. Endocrinol. 4:201-208). Contransfection ofpCMV-LC and pCMV-HC resulted in 392 ng/ml of MAbs determined by a MABELISA whereas transfection of pCMV-LC-emcvIRES-HC resulted in 185 ng/mlof MAb.

The CMV-LC-emcv-HC cassette was inserted in a retroviral vector based onthe Moloney murine leukemia virus (MLV), creatingpL-CMV-LC-emcvIRES-HC-RN-BG. LMH cells (see also Kawaguchi et al. (1987)“Establishment and characterization of a chicken hepatocellularcarcinoma cell line, LMH” Cancer Res. 47:4460-4464), the parent line ofLMH/2a, were used as target cells because they are not neomycinresistant. LMH cells were transduced with the L-CMV-LC-emcvIRES-HC-RN-BGretroviral vector and selected with neomycin and passaged for severalweeks. LMH cells were separately transduced and neomycin selected withthe parent MLV vector, LXRN. Media from LXRN cells were negative forMAb, whereas media from the L-CMV-LC-emcvIRES-HC-RN-BG-transduced cellscontained 22 ng/ml of MAb.

All documents (e.g., U.S. patents, U.S. patent applications,publications) cited in the above specification are herein incorporatedby reference. Various modifications and variations of the presentinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific preferred embodiments, itshould be understood that the invention as claimed should not be undulylimited to such specific embodiments. Indeed, various modifications ofthe described modes for carrying out the invention which are obvious tothose skilled in the art are intended to be within the scope of thefollowing claims.

1. A composition comprising interferon-α wherein the interferon isglycosylated with

wherein Gal=Galactose, NAcGlu=N-Acetyl-Glucosamine, andNAcGal=N-Acetyl-Galactosamine.
 2. The composition of claim 1 wherein thecomposition is a pharmaceutical composition.
 3. The composition of claim1 wherein the interferon is interferon α2.
 4. The composition of claim 1wherein the interferon is interferon α2b.
 5. The composition of claim 1wherein the glycosylation is O-linked.
 6. The composition of claim 1wherein the interferon comprises SEQ ID NO:
 30. 7. A compositioncomprising interferon-α wherein the interferon is glycosylated withGal-Gal-NAcGal-, wherein Gal=Galactose, andNAcGal=N-Acetyl-Galactosamine.
 8. The composition of claim 7 wherein thecomposition is a pharmaceutical composition.
 9. The composition of claim7 wherein the interferon is interferon α2.
 10. The composition of claim7 wherein the interferon is interferon α2b.
 11. The composition of claim7 wherein the glycosylation is O-linked.
 12. The composition of claim 7wherein the interferon comprises SEQ ID NO:
 30. 13. A compositioncomprising interferon-α wherein the interferon is glycosylated with

wherein Gal=Galactose, NAcGal=N-Acetyl-Galactosamine, and SA=SialicAcid.
 14. The composition of claim 13 wherein the composition is apharmaceutical composition.
 15. The composition of claim 13 wherein theinterferon is interferon α2.
 16. The composition of claim 13 wherein theinterferon is interferon α2b.
 17. The composition of claim 13 whereinthe glycosylation is O-linked.
 18. The composition of claim 13 whereinthe interferon comprises SEQ ID NO: 30.